BBI Products Catalogue

Research Product Catalogue

2012/13

ww.buybbi.com

Contents

1 Buy products online at www.buybbi.com • [email protected]

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12

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31

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1. About BBI

2. Introduction to Gold Labelling

3. Colloids

4. Gold Colloid Products and Pricing

5. Silver Colloid Products and Pricing

6. Microarray Gold and Silver Products and Pricing

8. High OD Gold Colloid

7. How to Choose a Gold Conjugate

12. Getting the Best from Immunogold Labelling

9. EM Grade Gold Conjugates

10. Gold Conjugate Products and Pricing

11. GoldLink – Rapid Conjugation Kit

13. Gold Stains Products and Pricing

14. Silver Enhancing Products and Pricing

15. Unicryl Products and Pricing

16. Ancillary Products and Pricing

17. How to Order

18. BBI Distributors

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BBInternational is a leading manufacturer of high quality gold and silver nanoparticles and reagents for a range of research, diagnostic and nanotechnology applications. Our products, which are manufactured to a proprietary technique, are subject to a number of stringent quality control measures.

High Quality ProductsOur products are manufactured and tested with the highest possible emphasis on reliability and reproducibility. Every product is rigorously tested with the most stringent quality assurance procedures. Because of this policy we now offer the widest range of top quality colloidal gold and silver nanoparticles, reagents and associated products world-wide.

About BBI

[email protected] • Buy products online at www.buybbi.com 2

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Technical supportWe offer a high level of technical support. Our in-house team is always available to offer help and troubleshooting advice, on the use of nanoparticles and conjugates. To give greater assistance in choosing and using our products, we have included a section on the choice of gold conjugate depending on application and requirements. We also have a range of additional technical information, including technical booklets and protocols. This information is available on request. Additionally our Technical Support department can be contacted on +44 (0) 29 2074 7232 or e-mail at [email protected]

CollaborationWe collaborate with many laboratories around the world. In our laboratories we have a number of ongoing programmes using nanoparticles and reagents in various applications for the development and application of new products. Many of the images and data that appear in our literature have arisen from these joint collaborations. We welcome any requests for collaboration on new techniques, products or applications.


Products OverviewGold Colloid BBI’s unique recipe gold colloid (colloidal gold) has been used for over 25 years due to its superior quality and performance. The optical properties of gold at the nanoscale means there is a variation in colour from red to pink to orange depending on nanoparticle size, a property that can be successfully exploited in a range of applications. BBI’s colloidal gold offers excellent stability and sensitivity characteristics, and as the market has grown to expect higher levels of sensitivity, gold has increasingly become regarded as an extremely reliable raw material in providing an accurate visual signal.

Silver Colloid The use of silver can potentially lead to improved sensitivity where silver nanoparticles demonstrate a stronger, more distinctive SPR spectrum than the corresponding gold particles. Use of silver in place of gold can potentially result in a more defi ned wavelength shift, and broadens the range of optical properties available – an advantage for applications where multiplexing is needed.

Gold Conjugates A colloidal gold conjugate consists of a suspension of gold particles coated with a selected protein or macromolecule (such as an antibody). BBI has a range of EM grade products, and LM grade products, that combine different antibodies on the range of particles predominantly, from 2, 5, 10, 15, 20, 30 & 40nm.

Custom Conjugation ServiceOur many years of expertise in manufacturing and using gold and silver nanoparticle reagents has enabled us to provide a comprehensive service for custom conjugation of products not listed in the Catalogue. We have the capability to work with a range of proteins, including antibodies and antigens as well as the more complex area of nucleic acid conjugation. We also offer conjugation to other types of particle including; latex, carbon and polymers.

High OD Gold ColloidBBI’s high OD gold can be used to provide dense uniform mono-layers of gold particles on substrates and can also simplify the conjugation procedure by removing the concentration steps. This saves time, reduces costs and ensures waste is minimised.

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High OD Gold Colloid can be custom made to your specifi c requirements. Whether you require 40nm particles at 40OD, 150nm particles at 150OD or 250nm particles at 250OD, our highly experienced technical team are able to help.

Products and Services for DiagnosticsIn addition to the research products listed in this catalogue we also offer end to end OEM diagnostic services including:

• Bulk supply of gold and silver colloid and conjugates• Custom conjugation of proteins and oligonucleotides• Research and development of rapid diagnostic tests• Rapid molecular diagnostic platforms • Contract manufacture• Assay manufacturing machinery

If you would like to discuss ways in which BBInternational can help you reach you rapid assay development goals, please contact us at [email protected] or visit our web site at www.bbigold.com

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HistoryOver 50 years ago fl uorescent labels were conjugated with antibodies for the fi rst microscopical detection of antigens in tissues. In 1970 enzyme labels fi rst provided a permanent coloured stain with higher resolution and greater stability than fl uorescent signals. With the advent of gold labels in 1971, sensitive high resolution immunocytochemistry became possible in the electron microscope for the fi rst time. The subsequent development of silver enhancing methods then allowed gold labels to provide a highly specifi c and sensitive method for visualising antigens in light microscopy. Since their introduction to microscopy, gold labels have also become recognised as very important tools for detection and quantitation of proteins, antigens, and nucleic acids when used with other techniques such as blotting, fl ow cytometry, hybridisation, and fi ngerprint identifi cation.

A very important use for gold conjugates has emerged in their incorporation into rapid test immunoassays. In these techniques the unique red colour of the accumulated gold label, when observed by lateral or transverse fl ow along a membrane on which an antigen is captured, or by observation of the red colour intensity in solution, provides an extremely sensitive method for detecting sub nanogram quantities of proteins in solution.

Gold ProbesGold labels act as markers for molecules that are otherwise invisible by eye or through other detection systems. A colloidal gold conjugate consists of a suspension of gold particles coated with a selected protein or macromolecule (such as an antibody). The gold particles may be manufactured to any chosen size from 2-500nm. This gold probe detection system, when incubated with a specifi c target, such as in a tissue section, will reveal the target through the visibility of the gold particles themselves.

Under an electron microscope the particles are visible without further treatment. Under a light microscope, however, the gold particles are made visible through a short and simple silver enhancing procedure.

For detection by eye, gold particles will also reveal immobilised protein on a solid phase such as a blotting membrane through the accumulated red colour of the gold sol. Silver enhancement of this gold precipitate also gives further sensitivity of detection.

ConjugationThe conjugation of selected proteins to gold particles depends upon at least three physical phenomena:

a) Charge attraction of the negative gold particle to positively charged protein

b) Hydrophobic adsorption of the protein to the gold particle surface

c) Binding of the gold to sulphur (dative binding) where this may exist within the structure of the macromolecule.

Adsorption of proteins to gold colloid.

Gold may be conjugated to a wide variety of molecules including proteins (e.g. antibodies), polypeptides, carbohydrates, polymers, polysaccharides, enzymes and nucleic acids. The conditions under which a gold conjugate is manufactured drastically affect its performance and stability.

Introduction to Gold Labelling

oteinsPro

Gold Colloid

StabilisedGold Colloid

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Why Gold?There are several advantages in choosing gold particles as markers for microscopic and non-microscopic identifi cation of target molecules.

The choice of particle size makes it possible to study antigens microscopically over a wide range of magnifi cations and provides the opportunity for multiple labelling of antigens on tissue sections. In the electron microscope labelling with gold particles is completely unequivocal. The label cannot easily be mistaken for any other tissue structure and is either present or not present. The resolution of this labelling in both Electron Microscopy and Light Microscopy is thus higher than with any other method. While fl uorescent labels and enzyme based colour reactions may fade with time and light exposure, gold particles do not disappear but give a permanent label.

Because of the particulate nature of the gold label, quantitation in the electron microscope is possible, even for more than one antigen by multiple labelling with different particle sizes.

In the light microscope, the intense brown/black stain produced by silver enhancement of the gold signal has been shown to give a greater overall sensitivity and far greater resolution than other immunocytochemical methods and tissues can be counterstained with all the usual staining procedures.

Unlike some enzyme based detection systems; gold probes are essentially non toxic, safe and easy to use. The good stability of a well made gold conjugate gives the product a long shelf life whether frozen or stored at 2-8°C.

For the staining of proteins immobilised onto membranes, gold probes have unsurpassed detection sensitivity and resolution.

Finally, and perhaps surprisingly, gold probes are inexpensive in their application and their high sensitivity often allows valuable primary antibodies to be diluted signifi cantly further than with other systems.

What Determines High Quality?Several essential features must apply to gold probes if they are to be considered reliable high quality reagents for experimental and diagnostic research.

Antibodies and ProteinsTo begin with the raw materials, all antibodies and proteins used for conjugation are recommended to be affi nity purifi ed and of the highest quality. They must possess very strong affi nity for the specifi c antigen and have high avidity to withstand severe incubation and washing conditions. Cross reactivity must be the lowest possible. In order to ensure consistent and reliable results, BBInternational uses only the highest quality antibodies and proteins for reagent manufacture.

BBInternational has perfected a proprietary technique for the manufacture of mono-disperse gold and silver colloids, to pre determined sizes with a coeffi cient of variation (CV) of less than 8% of particle size. This ensures our gold conjugates are consistent from batch to batch and give the same high performance time after time. In addition each batch is stringently quality controlled to ensure our high specifi cations are adhered to.

Sensitivity and StabilitySensitivity is all important in immunolabelling for detection of low levels of antigens in cells and tissues. In order for a gold probe to produce a strong specifi c signal together with a low background the conjugation of antibody to gold particle must be complete and stable under a variety of incubating conditions. The gold particle must have the minimum effect on the activity of the antibody to which it is conjugated but be strongly enough absorbed to the surface to remain stable for years. For long term storage conjugates are made using glycerol based making it possible to freeze a gold conjugate without loss of the antibody activity. All standard BBInternational gold conjugates may be frozen and stored over long time intervals without deterioration.

Introduction to Gold Labelling Ab

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ClusteringFor electron microscopy a very low level of clustering is important, especially if quantitation is to be performed or high quality micrographs are required. Clustering indicates a poor absorption of protein with bridging between gold particles. Heavily clustered conjugates have low stability and poor sensitivity. At the very least gold conjugates should have greater than 85% singlets.

All BBInternational EM Grade gold conjugates are made to this high specifi cation.

Quality AssuranceRigorous testing and fully documented QC protocols ensure that all of our products meet tight specifi cations, thus ensuring the highest possible consistency and performance. It is this focus on quality control, coupled with our unique manufacturing process that has resulted in BBInternational colloids being recognised as the ‘Gold Standard’ within the Life Science and Lateral Flow diagnostics industry.

Introduction to Gold Labelling

Antibody Purifi cation

Determine Antibody Titre

Manufacture and Concentrate Conjugate

Binding Analysis

Column Chromatography

BBI are experts in creative conjugation. Let us conjugate your antibody to gold colloid.

Talk to us about your requirements

Tel: +44 (0) 29 2074 7232

[email protected]

• 3ml fi nished conjugate• Technical advice and ongoing support

CREATIVE CONJUGATION SERVICE

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Gold ColloidsThe full range of BBI high quality gold conjugates is listed in this Catalogue. However, we recognise that there may be occasions when customers may need to use gold conjugates other than those listed here. Solutions of unconjugated colloidal gold sols are available from BBI in a wide range of particle sizes. These colloids may be conjugated to antibodies, proteins, or many types of macromolecule for binding and reaction labelling studies.

The same high quality manufacturing principles are used for the production of these gold sols as those used in the preparation of BBI gold conjugates listed in this Catalogue. Colloids are made with a very narrow size distribution, the actual size being measured by electron microscopy and provided on the data sheet with each product. The colloids are provided in sterile containers ready for conjugation. They will remain stable until the use by date if stored unopened at 4°C. Do not freeze.

Which Gold Colloid to Choose?The complete range of particle sizes of BBI gold colloids is given below. The choice of particle size depends on the specifi c application. Please also refer to the preceding Sections for applications in EM, LM or Blotting applications.

a) Light MicroscopyFor light microscopy, in conjunction with silver enhancing, the smaller sizes are recommended (2nm and 5nm).

b) Electron MicroscopyFor electron microscopy any particle size may be used, the smaller particles giving the highest labelling effi ciency. The magnifi cation required for the EM study determines the particle size to be used. Smaller particles may be silver enhanced for viewing at lower magnifi cations.

For scanning electron microscopy 20nm, 30nm or 40nm may be used together with backscattered electron imaging. However, smaller particles will give a higher labelling intensity and may be silver enhanced.

c) BlottingFor blotting applications the larger particles give the most visible stain without silver enhancing. Larger particles may produce more steric hindrance to the labelling, however. The choice lies between 20-40nm for many applications. For use with the BBI Blotting Silver Enhancing Kit any particle size may be used, but again smaller particles will give the highest labelling intensity. For silver enhancing we recommend 2nm or 5nm gold particles.

Unit SizeBBI gold colloids are available in 20ml, 100ml and 500ml volumes in polycarbonate bottles. Please indicate the required volume when ordering. Please refer to the current price list from your local distributor.

BBI gold colloids of 20nm and above are supplied at optical density 1.0 measured at 520nm. 5, 10, and 15nm are supplied at approximately OD0.8. 2nm colloids are approximately OD0.02 at 400nm. Colloids contain 0.01% concentration of HAuCl, except EM.GC2 which has a concentration of 0.001%.

Bulk Purchase of Gold ColloidsLarger volumes of gold colloids of any chosen particle size can be supplied for bulk purchase. Please contact us for details by emailing [email protected].

Colloids Ab

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Gold Colloid Supporting Data Particle Diameter

(nm)No. particles

per mlMass of gold

per ml (g)Zeta Potential

(mV)

0

0.2

0 40.4

0.6

0.8

1

1.2

430 450 470 490 510 530 550 570 590 610

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Wavelength (nm)

Wavelengthaa scans (600-450nm) of Gold Colloid Life Science Starter Kit

5nm

10nm

20nm

40nm

(nm)( )

5nm = 517.010nm = 518.22020nm 52= 5244.8840nm = 526.4

WavelengthPeak

0

0.2

0.4

0.6

0.8

1

1.2

1.4

430 450 470 490 510 530 550 570 590 610

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Wavelength (nm)

Wavelength aa scans (600-450nm) of Gold Colloid Diagnostic Starter Kit

20nm

40nm

60nm

80nm

Peak Wavength(nm)

20nm = 524.840nm = 526.460nm = 535.480nm = 550.0

2 1.50E+14 1.21-05 -34.1

5 5.00E+13 6.32-05 -38.6

10 5.70E+12 5.76-05 -38.4

15 1.40E+12 4.77-05 -42.8

20 7.00E+11 5.66-05 -41.4

30 2.00E+11 5.46-05 Data currently not available

40 9.00E+10 5.82-05 -44.0

50 4.50E+10 5.68-05 Data currently not available

60 2.60E+10 5.68-05 -50.2

80 1.10E+10 5.69-05 -52.8

100 5.60E+09 5.66-05 -56.3

150 1.66E+09 5.66-05 -60.5

200 7.00E+08 5.66-05 -56.1

250 3.60E+08 5.66-05 -66.7

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Gold Colloid ApplicationsToday’s world is connected at the touch of a button and we live in a society that thrives on knowledge. If that knowledge can be passed to health experts, they then have the opportunity to complete fast, frequent, reliable and cost effective diagnosis to help deliver effective and effi cient treatment to patients.

The table below identifi es various areas of application for gold nanoparticles and the functions that these particles play within this area of research. These range from the original design uses such as immunohistochemistry and lateral fl ow diagnostics, to drug delivery, chemical sensing and even forensic science and textile printing and development.

Nanoparticle SizeRange

Areas of Use Function

Small(2nm-15nm)

Medium(20nm-60nm)

Large(80nm-250nm)

Immunohistochemistry,Light Microscopy,High Magnifi cation TEM,Drug Delivery, Biomarkers,Gas Sensors, Coatings

Lateral Flow Assays,TEM, SEM, Environmental Detection and Purifi cation, Data Storage, Drug Delivery, Biomarkers, SERS, Photo-thermolysis, CatalysisChemical Sensors, DNA Detection

Flow Cytometry, Printed Text, Conductive Films, SERS, Forensic Science, Electronic Device Manufacture, SERS, Optical Mammography

• Silver enhanced antibody detections within cells• Solvent suspensions in gas detection• Decorative coatings in paint and textile

industries• Thin fi lm substrate coatings for semi-conductor

manufacture or electrochemical devices• Tumour targeting in cancer for drug delivery or

identifi cation of substrates

• Mercury detection and removal from contaminated water

• Fluorescent probes• Molecular imaging agents• Colorimetric competition assays• Glucose biosensors• Single nucleotide polymorphism detection• Tumour cell targeting and imaging• Chemical reduction catalysis• Creation of C-C bonds and oxidation of organic

substances

• Detection of CD4 cells using fl ow cytometry• Fuel cell effi ciency• Support of silk fi broin fi bres for use in the textile

industry• Direct printing of gold particles onto paper and

fabric• Detection of biomarkers in cells for imaging and

sensing purposes

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Optical Properties of Colloidal Silver20nm silver colloid shows a pronounced absorption peak around the 400nm wavelength of light. This is in contrast to gold colloid which exhibits an absorption peak around the 520nm wavelength. This results in silver colloid possessing an intense yellow colour as opposed to the characteristic ruby red colour of gold colloid and the black colour produced by platinum colloid.

These differing colours allow the labelling of different analytes with these various noble metal nanoparticles. BBI has the capacity and experience to produce high volumes of silver colloid at exceptionally high quality standards, allowing multiplexing applications within lateral fl ow tests.

BBI’s silver colloid is available in 20, 40, 60 and 80nm particle sizes. The colloidal silver is made according to BBI’s unique recipe and offers superb quality and performance. The colloid is produced by a wet chemical method and uses citrate as a stabilising agent. The particles possess a citrate shell which gives them a net negative charge. This allows the nanoparticles to repel each other and stay in solution. For larger particle sizes (60 and 80nm) the surface charge is unable to prevent the particles aggregating together and settling at the bottom of their container. However, the particles can be re-suspended into solution simply by inverting the bottle and gentle shaking. BBI has developed a unique recipe silver colloid; allowing for precise control of the particle size, monodispersity and large batch sizes of up to 56L, at standard concentration.

In summary, the interesting features of colloidal silver such as its antibacterial effects and unique optical properties, along with recent advances in fi elds such as SERS and electronics manufacture, make it a colloid with a multitude of uses. In order to get the best results from a silver colloid, researchers and engineers need a high quality, consistent and monodisperse particle. BBI’s silver colloid offers superb performance with a large batch size and is suitable for use in a wide range of applications.

Characteristics of BBI Silver Colloid

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

320 350 375 400 425 450 475 500 525 550 575 600

Abs

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Wavelength (nm)

80nm

60nm

40nm

20nm

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Additional NotesThe colloids manufactured by BBI are for the purpose of conjugation to macromolecules, (antibodies, antigens, proteins, etc.), and BBI does not guarantee the suitability for any other purpose. Optimising the conjugation of proteins is a complex, and for the inexperienced, a time consuming process. BBI have many years of experience in this respect, and offer a custom conjugation service, which will give you an optimised conjugation within a short time frame. Although the cost of this service is higher than a bottle of gold, the development time saved will offset this cost. To discuss your requirements, please contact our custom conjugation department, (email: [email protected]). It should also be noted that not all proteins are suitable for conjugation to gold colloid. If you are unsure about the suitability of these colloids for your purpose, please contact us at the above email address and we will be pleased to discuss your application.

If a bottle of colloid is left undisturbed for a period of time, the colloid will start to settle out and a ring of gold particles will appear in the bottom of the bottle. Again this is normal and can be easily resuspended by inversion of the bottle several times.

Colloids should be stored correctly to prevent them from precipitating. They should be stored between 2-8°C, although for short term storage the is no problem with room temperature. On no account should the colloid be allowed to freeze. Even partial freezing of the colloid will cause it to precipitate out of solution. This results in a clear solution with black lumps of precipitate in the bottom of the bottle. Therefore when storing the colloid do not place it close to the plate at the back of a fridge, or directly in front of the cold air in a cold room. Colloids damaged in this way will not be replaced as part of the guarantee.

Bottles should only be opened in a clean room, preferably in a laminar fl ow cabinet. Remove the colloid by pouring out what you need. Never place a pipette or anything in the bottle that can introduce contamination. pH of colloids can be adjusted by using 1% or 0.1% HCl or NaOH solutions. Note that at extremes of pH, the colloid will collapse. Never add high molarity buffer salts to the colloid, without checking a small volume fi rst. Colloid will turn purple / blue on contact with high molarity salts, and precipitate out of solution. Colloid damaged in this way will not be replaced as part of the guarantee.

Colloids will keep well for many months unopened and are guaranteed until the expiry date unopened. Once opened, they may start to deteriorate and eventually precipitate. For this reason purchase only what you will need to use immediately, and discard any remaining colloid one month after opening. Purchasers of large volumes of colloid may like to receive their colloids packaged in convenient sized containers. Please contact our technical department to discuss your requirements.

Colloids


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Gold ColloidsBBI’s unique recipe gold colloid has been market leading for over 25 years due to its superior quality, consistency and performance, offering excellent stability and sensitivity characteristics. There are several advantages of choosing BBI’s gold colloid:

• Lowest available coeffi cient of variation (CV)• The choice of particle size makes it possible to

study antigens microscopically• Allows multiple labelling of antigens on tissue

sections• Labelling is completely unequivocal• Provides a permanent label• Quantitation under the electron microscope is

possible due to its particulate nature• The optical properties mean there is a variation

in colour from red to pink to orange depending on particle size, a property that can be successfully exploited in a range of applications

• Known to be catalytically active for a range of commercially important reactions

Sizes

Product 20ml 100ml 500ml

Code Size Code

EMGC2 2nm EMGC5 5nmEMGC10 10nmEMGC15 15nmEMGC20 20nmEMGC30 30nmEMGC40 40nm

EMGC50 50nmEMGC60 60nmEMGC80 80nmEMGC100 100nmEMGC150 150nmEMGC200 200nmEMGC250 250nm

Size

EMMGCC500EMMGCC600 EMMGCC800EMMGCC1000 EMMGCC1550 EMMGCC2000 EMMGCC2550

Gold Colloid Starter PacksA wide range of particle sizes can be conjugated to proteins and macromolecules, and fi rst time gold users or those trying out a new process may be unsure of which size to purchase. Our gold colloid starter packs provide a range of particle sizes allowing a comprehensive evaluation.

Product Code

Gold Colloid 5nm/10nm/20nm/40nm GCIKITLIFE 20 ml of each £131.00

Gold Colloid 20nm/40nm/60nm/80nm GCIKITDIAG 20 ml of each £131.00

Gold Colloid 20nm/40nm/60nm/80nm GCKITDIAGPLUS 100 ml of each £440.00

Volume Price

1000 mll of eeach

20 ml oof eaach

20 ml oof eaach

£1331.000

£1331.000

£4440.000

Pricing


Gold Colloid £37.00 £146.00 £487.00

Array Gold Colloid See p.14

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Silver ColloidThe use of silver can potentially lead to improved sensitivity where silver nanoparticles demonstrate a stronger, more distinctive SPR spectrum than the corresponding gold particles. Use of silver in place of gold can potentially result in a more defi ned wavelength shift, and broadens the range of optical properties available – an advantage for applications where multiplexing is needed.

Product 20ml 100ml

Silver Colloid £37.00 £146.00 £487.00

Array Silver Colloid See p.14

500ml

£1146.000 £44877.000

Sizes

Code Size Code

EMSC20 20nm EMSC40 40nm

EMSC60 60nmEMSC80 80nm

Size

EMMSCC600EMMSCC800

600nmm800nmm

Pricing


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Microarray Gold & Silver ColloidIn the Microarray market, gold with its unique properties has emerged as an alternative to fl uorescence detection.

BBInternational’s Microarray gold is the ideal solution for overcoming many of the problems associated with fl uorescence detection methods, including low sensitivity, signal intensity, reduction due to photochemical bleaching, quenching and expensive image analysis systems.

Code Size Volume

ArrayGC2 2nm 20ml

ArrayGC5 5nm 20ml

ArrayGC10 10nm 20ml

ArrayGC15 15nm 20ml

ArrayGC20 20nm 20ml

ArrayGC30 30nm 20ml

ArrayGC40 40nm 20ml

ArrayGC50 50nm 20ml

ArrayGC60 60nm 20ml

ArrayGC80 80nm 20ml

ArrayGC100 100nm 20ml




220ml

220ml

220ml

220ml

220ml

220ml

220ml

220ml

220ml

220ml

220ml

220ml

220ml

220ml

Code Size Volume

ArraySC20 20nm 20ml

ArraySC40 40nm 20ml

ArraySC60 60nm 20ml

ArraySC80 80nm 20ml

220ml

220ml

220ml

220ml

Microarray Gold Colloid £110.00 Microarray Silver Colloid £110.00

Product 20ml

Key Features

• Highly Specifi c

• Reproducible

• Stable

• Highly Sensitive

• Reliable

• Perfectly Spherical Shapes (≤95 %)

• Low CV (<8%)

Pricing


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High OD gold can be used to provide dense uniform mono-layers of gold particles on substrates and can also simplify the conjugation procedure by removing the concentration steps. This saves time, reduces costs and ensures waste is minimised.

BBI’s high OD Gold Colloid can be custom made to your specifi c requirements. Whether you require 40nm particles at 40OD, 150nm particles at 150OD or 250nm particles at 250OD, our highly experienced technical team are able to help. Please enquire for pricing.

High OD Gold Colloid


Particle Diameter OD@520nm

No. Particles per ml

No. MolesParticle per ml

MolarParticle Concentration

(No. moles per L)

Mass of Gold per ml

(g)

Moles of Gold per litre

20 1 7.00E+11 1.1624E-12 1.1624E-09 5.66E-05 2.87E-04

20 5 3.50E+12 5.81202E-12 5.81202E-09 2.83E-04 1.44E-03

20 10 7.00E+12 1.1624E-11 1.1624E-08 5.66E-04 2.87E-03

20 15 1.05E+13 1.74361E-11 1.74361E-07 8.49E-04 4.31E-03

20 50 3.50E+13 5.81202E-11 5.81202E-07 2.83E-03 1.44E-02

20 100 7.00E+13 1.1624E-10 5.81202E-05 5.66E-03 2.87E-02

40 1 9.00E+10 1.49452E-13 1.49452E-10 5.82E-05 2.96E-04

40 5 4.50E+11 7.4726E-13 7.4726E-10 2.91E-04 1.48E-03

40 10 9.00E+11 1.49452E-12 1.49452E-09 5.82E-04 2.96E-03

40 15 1.35E+12 2.24178E-12 2.24178E-09 8.73E-04 4.43E-03

40 50 4.50E+12 7.4726E-12 7.4726E-09 2.91E-03 1.48E-02

40 100 9.00E+12 1.49452E-11 1.49452E-08 5.82E-03 2.96E-02

60 1 2.60E+10 4.3175E-14 4.3175E-11 5.68E-05 2.88E-04

60 1 1.30E+11 2.15875E-13 2.15875E-10 2.84E-04 1.44E-03

60 1 2.60E+11 4.3175E-13 4.3175E-10 5.68E-04 2.88E-03

60 1 3.90E+11 6.47625E-13 6.47625E-10 8.51E-04 4.32E-03

60 1 1.30E+12 2.15875E-12 2.15875E-09 2.84E-03 1.44E-02

60 1 2.60E+12 4.3175E-12 4.3175E-09 5.68E-03 2.88E-02

80 1 1.10E+10 1.82664E-14 1.82664E-11 5.69E-05 2.89E-04

80 1 5.50E+10 9.13318E-14 9.13318E-11 2.85E-04 1.44E-03

80 1 1.10E+11 1.82664E-13 1.82664E-10 5.69E-04 2.89E-03

80 1 1.65E+11 2.73995E-13 2.73995E-10 8.54E-04 4.33E-03

80 1 5.50E+11 9.13318E-13 9.13318E-10 2.85E-03 1.44E-02

80 1 1.10E+12 1.82664E-12 1.82664E-09 5.69E-03 2.89E-02

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The aim of an antibody incubation with a specimen, whether it be a section, cells in suspension, a tissue slice, a blotted membrane, or a rapid diagnostic test assay, is to achieve the most intense specifi c signal and the least non specifi c background possible. The next section; ‘Getting the Best from Immunogold Labelling’ section describes this in detail but the initial choice of gold conjugate is also important.

Direct or indirect labelling (antibodies)

Silver enhancing of direct or indirect labelling.

Gold conjugates may be used directly or indirectly to label antigens as indicated above. In the direct method the gold is conjugated to the primary antibody, whether monoclonal or polyclonal. This allows a single incubation to be performed and provides the simplest detection system. In the indirect method a primary unlabelled antibody is applied to the specimen to locate the antigen. This is followed by a gold labelled secondary that detects the primary antibody. This gold labelled secondary antibody is almost always an affi nity purifi ed polyclonal. In this way an amplifi cation of the signal is achieved, often up to 10x compared with a direct incubation, according to the particle size.

The choice of the most appropriate system depends upon the type of component to be detected and what binding proteins are available. Some guidelines on making this choice are as follows:

An extension of the indirect method may be used where there is no appropriate gold labelled secondary to match the primary antibody. For example if the unlabelled primary is from Pig and there is no matching gold labelled anti-Pig then an unlabelled anti-Pig (say Rabbit anti-Pig) may be used as a second step followed by a gold labelled third antibody (say Goat anti-Rabbit) that will detect the second antibody.

The indirect method is the most common for studying cells and tissues and avoids the need to label every primary antibody. It provides a universal method for detecting any primary antibody from the same primary species, i.e. all Rabbit antibodies may be detected by Goat anti-Rabbit gold conjugates, etc. The indirect method is longer than the direct method because of the need for two (or three) separate incubations but is the more widely used. The previous table indicates secondary gold labelled antibodies to be used with any chosen primary.

Absorption with serum proteinsAntibody gold conjugates are absorbed against certain species of serum proteins. This absorption reduces cross reactions to those species to an absolute minimum.

When to use F(ab’) fragments?In some applications background labelling may be a problem due to the attraction of the Fc region of the antibody gold conjugate to tissue components (called Fc receptors). Normally this is blocked by the simple application of normal serum prior to the fi rst antibody. If the problem persists, however, then gold labelled F(ab’) fragments of antibodies may be used. These conjugates are listed in each Section.

When to use antibodies, Protein A, Protein G or Protein A/G?Protein A (PAG), Protein G (PGG) and Protein A/G (AG) are all separate proteins and will mimic a secondary antibody by binding to the Fc part of a primary antibody. In some situations they may be used in place of the secondary antibody. Unlike secondary antibodies, however, it is believed that there is only a single binding site for these proteins on the primary lgG, They do not bind with great affi nity to IgM or lgA molecules.

How to choose a Gold Conjugate

Direct

Silver enhancing

Primaryantibody

ndarySeconantibody

Indirect


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The advantage of these proteins is that they provide a universal second step for a wide range of primary antibody species. They each bind with varying affi nities to IgG molecules of different species as shown in the table. However, if possible we would always recommend the selection of a species specifi c secondary antibody in preference to Protein A, G or A/G.

Second antibodies provide an amplifi ed signal compared with Protein A.

Use of F(ab’)2 fragments to avoid non specifi c labelling.

Streptavidin or Goat anti-Biotin?For the indirect detection of a biotinylated primary antibody or nucleic acid probe there is a choice of Streptavidin or Goat anti-Biotin gold conjugates. Historically streptavidin has been the most frequently used detector for biotin because of the very high affi nity constant between them. Most recently, however, Goat anti-Biotin has been shown to be a rather more sensitive detector of biotin compared to streptavidin when conjugated to gold particles. This is because of the relatively large molecular size of the antibiotin molecule (160,000 Daltons) compared to streptavidin (40,000 Daltons) and the distance between the binding of the gold on the Fc, from the binding region of the antibody F(ab’). This is especially so when using larger gold particles but becomes insignifi cant for 5nm and 1nm gold conjugates. On occasions where background from nonspecifi c attraction of goat antibodies may cause a problem streptavidin may provide a cleaner result. We recommend beginning with Goat anti-Biotin gold conjugates for the detection of biotin.

Cationic GoldCationic gold allows highly sensitive and discrete microscopical studies of anionic (i.e. negative) sites in cells and tissues. The gold conjugate is made by careful conjugation to poly-L-lysine, a highly positive amino acid chain. A simple one step incubation of sections with the diluted Cationic Gold conjugate reveals subcellular sites having net negative charge. The charge distribution can be seen at a range of magnifi cations by using Cationic Gold of different particle sizes.

Most cells of eukaryotic origin have a net negative surface charge from anionic plasma membrane components. This charge distribution is thought to be important in movement of various soluble macromolecules across cell walls. Thus the role of the surface charge in cellular behaviour through interaction with neighbouring cells can be studied. For in vitro and in vivo studies Cationic Gold may also be used effectively to study the uptake of anionic material by endocytosis.


Specific labelling

Whole 1gG

Fc receptor

G

PrimaryP

Antigen

Non Specific labellingN p


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(F(ab1))2fragment

No attraction

AntigennPrimary

SecondarySantibody:tib dagoldg

PrimaryProtein A:gold

How to Choose the Gold Particle SizeA wide range of particle sizes can be conjugated to proteins and macromolecules. In principle smaller gold particles produce a higher labelling intensity on the specimen. This is because of the reduced steric hindrance to antigen detection. Typically an antibody of 160,000 Daltons molecular weight will have a linear dimension of 8nm. Thus a 2nm gold particle, attached to the Fc region will hardly impede the antibody activity. A 20nm particle. however, while being more visible, will produce a greater steric hindrance by its proximity to the antigen binding region of the antibody. In addition the increased charge repulsion between larger particles reduces the number of labelled antibodies gaining access to the target antigen.

Different particle sizes are appropriate for different types of application as described below.

a) Light MicroscopyGold particles cannot easily be seen in the light microscope by bright fi eld viewing. They must thus be silver enhanced for greatest visibility. Small gold particles will give the greatest number of gold labelling on the specimen and each particle can then be subsequently silver enhanced for maximum visibility of signal. A choice can be made between 2nm and 5nm gold conjugates. The 5nm conjugates are used for most standard purposes and are recommended for initial studies. For even greater labelling intensity the 2nm gold conjugates are preferred. In both cases the gold signal is silver enhanced to make it visible in the light microscope.

b) Transmission Electron MicroscopyFor electron microscopy any particle size may be employed. For low magnifi cation work larger particles (e.g. 15 to 30nm) are more easily seen. For high magnifi cation studies the smaller particles (e.g. 5 to 10nm) are preferred. Again, smaller particles give higher labelling intensity and may be subsequently silver enhanced on the section to produce larger particles with this high labelling intensity. For those just beginning

immunogold labelling in the EM or performing studies over a range of magnifi cations, 10nm gold conjugates are recommended.

c) Scanning Electron MicroscopyThe resolution of the scanning electron microscope indicates that larger gold particles (e.g. >20nm) should be used for detection by back scattering electron signals. However, as mentioned above, a greater intensity of labelling is achieved with smaller gold particles, which can be subsequently silver enhanced with the LM/EM Silver Enhancing Kit. We would therefore recommend that a choice is made for SEM studies between 20 or 30nm gold conjugates for direct (un-enhanced) viewing, or 5nm conjugates which can be simply silver enhanced within minutes for observation at low magnifi cations. With the latter approach each gold particle grows spherically to approximately 30nm in 35 minutes. The reaction is simply stopped by washing in water.

d) BlottingFor blotting applications where the gold conjugates are to be applied to proteins immobilised on nitrocellulose membranes, the choice is between 2nm and 20nm EM Grade gold conjugates. For ultra high sensitivity 2nm conjugates are preferred in combination with silver enhancing. However the 20nm gold conjugates allow blotted proteins to be identifi ed on the immobilising membrane without silver enhancing. The 2nm and 5nm gold conjugates, because of their much reduced steric hindrance and greater particle density in solution, may be diluted much further to provide the same fi nal intensity compared with the 20nm conjugates. The 2nm and 5nm gold particles must be enhanced with the Blotting Silver Enhancing Kit.



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Gold labels are the most satisfactory method of labelling antigens for visualisation in the electron microscope. Since their introduction in 1971 there has been a vast growth in the number of applications in animal and plant biology as well as microbiology1. The main advantages of gold labelling lie in the high contrast and unequivocal nature of the label, the range of particle sizes suitable for different magnifi cations, the extreme sensitivity, and the stability of the signal.

Features of BBI EM Grade Immunogold ConjugatesDetailed datasheets available for download for all BBI gold reagents giving individual specifi cations for each product.

High SensitivityConjugates with particle sizes upto 20nm will detect 10pg of specifi c protein with each gold conjugate in immunoblotting tests (with silver enhancement).

Low ClusteringEach gold conjugate has greater than 85% singlets. Quantitation is possible by counting particle density. Micrographs form attractive publications.

Low Cross ReactivityAll BBI antibodies are affi nity purifi ed and of the highest quality. Cross reactivity with other species is minimal. Conjugates are further absorbed against specifi c serum proteins where indicated.

High Concentration Optical densities of gold conjugates at 520nm wavelength are measured as 3.0 for 5 and 10nm, 4.0 for 15 and 20nm or 5.0 for 30nm or 40nm EM Grade gold conjugates. This allows them to be used in a highly diluted form.

High Resolution Because of the discrete nature of the gold particle and the close adsorption of anti-body/binding protein to the surface, antigens are localised with very high resolution.

Narrow Particle Size Distribution The low coeffi cient of variation of the gold particles for each conjugate allows multiple labelling to be achieved for several antigens on the same specimen.

Long Shelf Life BBI Gold Conjugates are stable at 2-8°C for 12 months from date of manufacture. If the conjugate is stored frozen it will remain stable for longer. Conjugates stored frozen may be usable beyond their expiry date, but it is advisable to test sensitivity by immunoblotting fi rst.

Which Gold Conjugate?The appropriate gold conjugate may be selected according to the criteria described in How to Choose a Gold Conjugate. The primary antibody determines the choice of secondary gold conjugate. F(ab’) fragment gold conjugates may be appropriate where background staining is high.

Which Particle Size?For selection of particle size it is recommended that this is determined by the magnifi cation to be used in the EM as in the table. For the highest labelling intensity, however, the smaller gold particles are preferred. For 2nm and 5nm gold conjugates, a combination of gold labelling with silver enhancing will yield larger size particles with high labelling intensity. For those researchers beginning with immunogold labelling we recommend 10nm gold particle size for observation at a range of magnifi cations. Please feel welcome to contact us for discussion of your application at [email protected]

Types of EM SpecimensSpecimens may be prepared for labelling and examination in the electron microscope in a number of ways:

a) Post Embedding Labelling of SectionsThis is the most widely used technique for labelling antigens for EM examination. Here the tissue is fi xed and embedded and incubations performed on sections. Only the surface of the section is labelled. Ultrathin cryosections may be included in this category.

EM Grade Products


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b) Pre Embedding Labelling of Tissues and CellsIn this case tissues (e.g. cell cultures or cells in suspension) are labelled by incubation, usually after fi xation, and before embedding. This method is particularly appropriate for the identifi cation of antigenic sites on membranes of both isolated cells and micro organisms.

c) Negative Stain MethodThis is the most suitable method for identifi cation of antigens on micro-organisms dried onto an EM grid.

d) Replica TechniqueCell surface antigens on cultured cells may be examined with this method. Cells are fi xed and labelled before being replicated for study by EM.

e) Freeze FractureFreeze fracture is suitable for labelling cell membranes and for examination by thin sections or with replicas. With this technique it is possible to label plasma and intracellular membranes on isolated cells or within tissues.

Detection of surface antigens by immunolabelling and scanning electron microscopy.

Scanning Electron Microscopy (SEM)Large specimen areas may be examined by the SEM. Specimen surfaces are labelled by a pre-embedding method (b) and then prepared for SEM examination. Backscattered electrons provide an image of the gold particles on the specimen surface. Large gold particles (>20nm) may be imaged in the SEM with good resolution while the specimen surface is viewed with secondary electron emission. Alternatively, for higher labelling intensity, smaller (e.g. 5nm) gold conjugates are used and subsequently silver enhanced.

Methods of Use of EM Gold ConjugatesDetailed instructions for use of all BBI gold conjugates and silver reagents are given in the Technical Information Booklet.

Reference1 ‘Colloidal Gold& Principles, Methods and Applications”, Vols. 13, MA Hayat (Ed), Academic Press, 1989-1995*

EM Grade Products

tissues.

Primaryelectron beam

Backscatterelectrondetector

Secondaryelectrondetector

Gold particlesntSilver enhancemen

Cell surface

iissppllaayySSEEMM ddii

+–


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Gold ConjugatesA colloidal gold conjugate consists of a suspension of gold particles coated with a selected protein or macromolecule (such as an antibody). BBI has a range of EM grade products that combine different antibodies on the range of particles predominantly 2, 5, 10, 15, 20, 30 & 40nm. BBI’s gold conjugates provide the following benefi ts.

• Binding to BBI’s superior quality gold colloid ensures a very low level of clustering while maintaining high level stability and sensitivity

• Only highest quality antibodies and proteins are used, meaning consistently reliable products

• BBI’s gold conjugates average 95% singlet particle scommercially important reactions

Product 0.25ml 1ml

EM and all Conjugates £75.00 £224.00

(except EM. BSA)

EM. BSA Conjugates £53.00 £149.00

Microarray Conjugates £121.00 £424.00

Conjugate Key

£424.000

£1499.000

£22244.000

Bovine Serum AlbuminCationic Colloidal Gold Goat anti Mouse IgG & IgM Goat anti Guinea Pig IgGGoat anti Mouse IgG Fc (Gamma) Specifi cGoat anti Rabbit IgGGoat anti Rat IgGGoat F(ab)’2 anti Rabbit IgGGoat anti Mouse IgG (H+L)Protein ARabbit anti Goat IgGStreptavidin

BSA CGC GAFGAG GAMGARGATGFAR GMHLPAG RAGSTP

EM. BSA5 5nm

EM. BSA10 10nm

EM. CGC5 5nm

EM. CGC10 10nm

EM. CGC15 15nm

EM. CGC20 20nm

EM. GAF10 10nm

EM. GAG5 5nm

EM. GAG10 10nm

EM. GAM2 2nm

EM. GAM5 5nm

EM. GAM10 10nm

EM. GAR2 2nm

Product Code Size

EM. GAR5 5nm

EM. GAR10 10nm

EM. GAR15 15nm

EM. GAR20 20nm

EM. GAR30 30nm

EM. GAR40 40nm

EM. GAT10 10nm

EM. GFAR5 5nm

EM. GFAR10 10nm

EM. GMHL5 5nm

EM. GMHL10 10nm

EM. GMHL15 15nm

EM. GMHL20 20nm

Product Code Size

EM. PAG5 5nm

EM. PAG10 10nm

EM. PAG15 15nm

EM. PAG20 20nm

EM. RAG5 5nm

EM. RAG10 10nm

EM. RAG15 15nm

EM. RAG20 20nm

EM. STP2 2nm

EM. STP5 5nm

EM. STP10 10nm

EM.STP15 15nm

EM.STP20 20nm

Product Code Size

Best Seller

Best Seller

Best Seller

Best Seller

Best Seller

Best Seller


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Technical AssistanceIn addition to the choice of antibodies and particle size, the selection of the correct gold conjugate depends on the correct use of protocols for specimen preparation and incubation. At BBInternational we are happy to provide assistance in these methods and will gladly help in the choice of the correct gold conjugate for any specifi c application.

Please contact our Technical Support team on +44 (0) 29 2074 7232 or e-mail us on [email protected] with your enquiry.

In addition our web site www.buybbi.com has a FAQs section which includes the most common queries we have experienced.



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GoldLink™ kits are designed for early stage R&D, where users need to quickly explore the viability of a range of antibodies, before the conjugate is optimised on a larger scale. Users can now easily screen their antibodies of choice in-house in a cost effective way, before utilising BBI’s Custom Conjugation service to progress their development. This will streamline R&D time and ensure effi cient use of budget in the early process of establishing which antibodies are right for the product.

Features and Benefi ts· BBI’s gold colloid· Quick and easy process for antibody conjugation –

15 minutes· No antibody pre-treatment required· Single approach to antibody screening· Minimal user knowledge or experience required· Conjugate stability assured· Minimal antibody usage· Minimal antibody waste

Background and ProcedureThe kits, containing BBI’s unique recipe gold colloid, are stable to high salt buffers and are pre-activated for antibody labelling. The antibodies are attached to the functionalised gold by covalent conjugation through the lysine residues, which produce a stable, non-reversible linkage. No antibody pre-treatment is required. A single reaction buffer is used for conjugation of all antibodies. The process requires minimal antibody and does not need a purifi cation step, meaning time and cost savings.

Antibodies or proteins are covalently attached to ultra-stable BBI Goldlink Colloid at a very high OD, quickly and easily (see Figure 1 below). The hands-on time for the kit procedure is just 2 minutes and the conjugate is ready to use in 15 minutes. The Goldlink Colloid has a protective surface coat that can withstand the most extreme conditions (e.g. 1M NaCl or 2.5M NaOH at 70oC for >1 hour).

The GoldLink™ Colloid in the kits is supplied as a freeze dried mixture.

SensitivityThe level of conjugate sensitivity achieved through the use of a GoldLink™ kit is suitable for antibody screening during initial R&D (see graph below). For customers who want to achieve a higher level of sensitivity and/or a fully optimised conjugate, BBI also offers a Custom Conjugate service.

BBI will take your antibody of choice and using our long established, proven processes, optimise your conjugate, and then freeze your specifi c recipe before putting a commercial supply plan in place.

GoldLink

Dilute Antibody

Mix with reaction buffer

Add to freeze dried BBI Gold Colloid

Quench the reaction

Finished Conjugate


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Loading Comparison - BBI GoldLink™Kit vs. BBI Custom Conjugation

Purchase a KitThe following kits are available.

GoldLink

Product Code Reactions Gold Particle Size

Volume of Finished Conjugate

GLK3.40 3 40nm 50ul 20OD £125

GLK3.40 10 40nm 50ul 20OD £315

GLK3.40 1 40nm 1ml 20OD £315

440n

440n

440n

50

50

1

Price

££1225

££3115

££3115


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Detailed technical instructions for the correct use of all BBInternational research products can be found online. Each product has a corresponding datasheet available to download from www.buybbi.com that provides information on specifi cation and performance.

The primary aim of all immunolabelling procedures is to obtain a maximum specifi c signal with a minimum non specifi c background. This is true for EM sections, LM sections, and immunoblots on membranes. The results obtained in any immunolabelling procedure are limited mainly by the specimen preparation and incubation procedures. References given here describe a number of optimum conditions and protocols for immunolabelling in LM, EM and Blotting applications.

Specimen PreparationCorrect specimen preparation procedures are absolutely crucial for optimum labelling of antigens in cells and tissues. Methods commonly used for ultrastructural preservation in the EM or morphological preservation in the LM must usually be modifi ed to ensure that antigens are not only retained but also available for labelling. This often involves compromise with the structure but careful selection of preparation methods can yield excellent combinations of structural detail and immunochemical labelling.

For LM and EM studies cells and tissue sections may be studied by pre-embedding, post embedding, or cryotechniques. In addition whole cells may be

examined as cytospins, cell smears, cells in culture, and cells in suspension 1,2,3,4.

It should be recognised that each step of the preparation procedure is likely to incur some antigenic loss by extraction or alteration or masking. Cumulatively these losses may greatly reduce the overall labelling unless each step is optimised. This can usually only be achieved empirically but some guidelines are described below.

a) FixationCells and tissues may be fi xed for subsequent examination or may sometimes be labelled in the unfi xed state. Fixation of the tissue must also preserve antigenicity without compromise to structural characteristics. Fixatives either denature proteins by coagulation (e.g. acetone or methanol) or by forming additive cross linked compounds (e.g. aldehydes), or both. The resulting complexes inevitably differ from the unfi xed proteins in both their chemical and antigenic profi les. Each tissue requires its own fi xation protocol. For example, too much cross linking in a tissue with high protein density may mask many antigens. On the other hand a loose tissue with low protein content may disintegrate without adequate fi xation and antigens may simply be washed out. According to the antibodies employed for antigen detection, it may not be necessary to completely preserve the protein under investigation if at least the specifi c antigen is conserved. The types of fi xatives employed are shown in the table below.

Getting the best from Immunogold Labelling

Types of Fixatives Structure Antigen Preservation Suitability

Additive (cross linking)Formaldehyde ++ ++ TissuesGlutaraldehyde +++ + Tissues

CoagulativeAcetone + + CellsMethanol + + Cells

MixedFormal Acetone ++ + CellsPicric Acid ++ + Tissues

+++++

++ ++

++ ++

TTisssueesTTisssuees

CCellsCCells

CCellsTTisssuess


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For many tissues the best compromise is a mixture of formaldehyde (e.g. 2-4%) for rapid stabilising with low cross linking, and weak glutaraidehyde (e.g. 0.1%) for greater structural preservation. For cytological investigation a precipitating or coagulating fi x such as acetone or methanol may be preferred. Formal acetone has also been commonly used for fi xing cell preparations. In some cases for cell studies simple air drying may allow enough antigenic preservation for immunolabelling.

Post fi xation for electron microscopy has mostly involved the use of osmium tetroxide in order to preserve membrane components and provide image contrast. The introduction of osmium into tissue is not always desirable, however, and more recently tannic acid has been suggested as an alternative’.

Whatever method of fi xation is selected it must serve the dual function of retaining the essential structural and antigenic components of the tissue without introducing any material which may interfere with the labelling.

In some cases the introduction of heavy metals such as osmium or uranium into the tissue may cause increased non specifi c labelling and must be treated with caution.

b) WashingThorough washing of the tissues following fi xation is extremely important. It may be necessary to wash for at least as long as the tissue has been fi xed in order to remove excess aldehyde or other fi xation residues which may cause non specifi c labelling. In some cases quenching the tissue with ammonium chloride is performed to neutralise aldehyde groups. The wash is best performed in a buffer having a tonicity similar to the natural tissue state.

c) EmbeddingTissues may be embedded in paraffi n wax for LM, and resin for LM or EM studies. In either case the embedding should allow good preservation of the antigens without sacrifi ce of structural information. Generally there are two types of resin, epoxy resins which have an aromatic structure and are strongly cross linked, and acrylic resins which have lower cross linking. Epoxy resins are hydrophobic whereas acrylic resins may be hydrophobic or hydrophilic. The best structural preservation and stability is provided by epoxy resins while the best immunolabelling is usually achieved with acrylic resins. This is because acrylic resins cut in such a way as to reveal the proteins at the section surface and they also wet more easily, thus giving greater accessbility to the antibodies during subsequent incubations. Resins for EM and LM preparation are shown in the table below.


Types of Resin Cross Linking Hydrophilicity Structure/Stability

Epoxy

Araldite High Hydrophobic +++ +

Epon High Hydrophobic +++ +

Acrylic

UNICRYLTM - Hydrophilic ++ +++

Lowicryl - Hydrophilic ++ ++

LR White - Hydrophilic ++ ++

LR Gold - Hydrophilic ++ ++

Methacrylate - +/- Hydrophilic ++ ++

HHydroppho

HHydroppho

HHydropphili

HHydropphili

HHydropphili

HHydropphili

+//- HHyddro

++++

++++

+++

+++

+++

+++

+++

Antigen Preservation/ Access

++

++

+++++

+++

+++

+++

+++


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For the optimum results a compromise must be reached and the usual choice is of a polar (hydrophilic) resin with moderate cross linking. A popular acrylic resin formulation is given by UNICRYLTM, which gives excellent immunolabelling characteristics together with a high degree of stability and structural preservation. UNICRYLTM, in common with many other acrylic resins, also allows the embedding procedure to be performed at low temperatures. This ensures that vital components are not extracted during dehydration and that no excessive temperature rise occurs during polymerisation which may damage the tissue antigens,

d) BlottingFor proteins blotted onto membranes by Western blotting or dot blotting it is important to ensure complete transfer of the proteins to the membrane. Membranes must then be blocked with neutral proteins or surfactants (e.g. BSA or Tween20®) to fi ll unoccupied sites and prevent non specifi c background labelling. The membranes are then incubated in a similar fashion to tissue sections. The complete protein band/spot pattern may fi rst be revealed by immersing the membrane in a solution of PROTOGOLD® which stains all proteins with colloidal gold, yielding a red stain. This gives an indication of the location of all protein bands. A second identical membrane may then be incubated with the appropriate specifi c antibodies for identifi cation of specifi c proteins immobilised on the membrane. An incubation of the membrane with gold labelled second antibodies will reveal the presence of specifi c protein bands by a visible red colour where the gold particles accumulate. In order to achieve the best signal with the least background thorough washing is important after each step.

IncubationsIn order to achieve the highest possible specifi c signal with the lowest possible non specifi c background it is important to be aware of all the factors in both specimen preparation and in subsequent incubations which can affect these results. The fi gure above indicates some of the most common sources of non specifi c background that can be identifi ed by the use of controls and successfully eliminated. These factors

apply for all types of tissues, whether for EM or LM, and for immunoblots on membranes.

a) Primary antibodyThe primary antibody should be of high titre and of the highest specifi c purity to allow the greatest possible dilution. Cross reactivity must be low, especially with the sample tissue. A low quality primary antibody is the greatest cause of low specifi c signal and high background during the labelling procedure. The buffer composition and pH is important for the incubations. A normal TBS or PBS buffer is usually chosen with suitable additions to maintain low background. The specimen must be washed thoroughly with buffer between incubations.

(b) Secondary antibodyA high quality second antibody is essential to label the primary specifi cally and with low background. BBInternational gold conjugates are affi nity purifi ed and of the highest quality. They are provided suspended in Tris or Phosphate Buffered Saline for microscopical or immunoblotting use, with sodium azide preservative and are stable for years under the correct storage conditions. This is because our gold conjugates contain no free antibody, all antibodies being securely adsorbed at the surface of the gold. In addition the very high proportion of singlets ensures that clusters will not form in time during storage.

Their high titre and purity means that for incubations they may be used highly diluted (typically 1/100 to 1/400) in the typical incubating buffer shown below, so reducing non specifi c background whilst achieving a high signal intensity. Even at high dilutions (1/1000 or more) the conjugates are extremely stable and may be left for long term incubations.



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(c) DilutionsWhen establishing a new protocol it is necessary to determine the optimum concentrations of both the primary and the gold labelled secondary antibodies. This is done by fi rst incubating separate sections with various dilutions of the primary antibody over an appropriate range of values, e.g. 1/100 to 1/10,000. The various primary incubations are then followed by second incubations, using a constant dilution such as 1/100 of the gold labelled secondary antibody. The dilution of primary antibody giving the optimum signal and background is thus determined. The procedure is then repeated, this time using the determined dilution of the primary as a constant, and incubating with a selected range of dilution values of the gold conjugate, e.g. 1/10 to 1/400. Observation of the second set of results will indicate the choice of dilution of conjugate that should be used in further experiments. With both primary and secondary incubations a greater sensitivity may also be achieved by agitating the sample or fl ushing the reagents, so bringing fresh solution continuously to the target proteins at the tissue surface. If the antibody solutions are tolerant the incubations may also be performed at slightly elevated temperatures (up to 37°C).

d) BlockingNon specifi c reactive sites on tissues and cell surfaces as well as unoccupied sites within blotting membranes may need blocking before antibodies are applied to the specimens. The causes of non specifi c labelling may arise from sources shown in the table. Each background source will need its own blocking procedure either before or during the antibody incubations as shown in the table and as described in the Technical Information section. BBInternational can supply blocking reagents.

A typical incubating buffer suitable for both LM and EM incubations, which provides the components for eliminating the above sources of background may be as shown below. This buffer may be used for each step of the incubation protocol, followed by washing in water after the fi nal step.

Serum cannot be used in conjunction with Protein A, Protein G or Protein AG gold conjugates since these bind IgG molecules.

Select serum corresponding to the host of the gold labelled antibody. In this example goat is selected because the conjugate being used is Goat anti-Rabbit.

e) ControlsIn order to determine if a signal is genuine and to differentiate it from background labelling, both positive and negative controls must be used. These are simple to perform and should always be included in any staining protocol. Negative controls would typically include the following:

• Omit the primary antibody

• Use a non specifi c primary antibody of the same species

• Absorb the primary antibody with antigen before incubation

• Use a non specifi c second antibody

These negative controls will help identify the source of any non specifi c background. A good positive control using a specimen with high antigen content will test the whole labelling system. Full instructions for the use of controls are given in our Technical Instruction Booklet.

For LM, EM and Blotting applications the correct use of silver enhancing procedures is also important to maximise the signal and to avoid non specifi c background.

The technical Information online at www.buybbi.com gives full details of basic protocols, dilutions, buffers, blocking reagents, controls, trouble shooting and recommended reagents for many applications to help achieve the best from immunogold labelling.



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PROTOGOLDTM PROTOGOLDTM colloidal gold sol has been specially developed to give intense staining of proteins blotted onto membranes by dot blots or by electrophoretic gels (Western blotting). The gold particles are negatively charged and bind selectively to the blotted proteins with very low background absorption on the membrane. The specifi c staining of proteins occurs primarily by hydrophobic and ionic interactions.

The proteins stain dark red through the accumulation of gold particles on the membrane and the method produces a sensitivity greatly in excess of Coomassie blue or silver staining in gels with no fading of the signal. When used in conjunction with the BBI BL Silver Enhancing Kit a further enhancement of the signal by 10 to 100x is achieved.

Why Stain Membrane Blots?Staining of proteins on gels has been an established technique for many years. For the identifi cation of individual proteins in a gel, however, and for permanent storage, proteins must be transferred by blotting to a membrane such as nitrocellulose or PVDF. Individual protein bands can then be identifi ed by immunoblotting using specifi c primary antibodies followed by BBI secondary gold labelled antibodies.

Three signifi cant problems arise when trying to compare the total protein stain on a gel with an individual specifi c protein band on a membrane.

a) Transfer of the proteins from gel to membrane may not be the same for each protein band since the very nature of the electrophoresis method differentiates proteins by their size, charge and mobility.

b) Distortion between the gel and the membrane may occur so that matching proteins on the gel with individual proteins on the membrane may be diffi cult.

c) Handling and permanent storage of a gel, together with quantitative estimation of protein bands is diffi cult.

For these reasons it is sensible to compare individual specifi c proteins stained by immunoblotting on a membrane with total proteins stained on a duplicate membrane with PROTOGOLDTM. The method is extremely easy and results are very rapid.

By combining total protein staining with PROTOGOLDTM and specifi c immunostaining of individual protein bands with an enzyme labelled antibody (e.g. alkaline phosphatase) both signals can be achieved on the same membrane.

Features of PROTOGOLDTM

Fast Results Proteins are stained in minutes.

High Sensitivity Less than 1pg of protein is detectable (with silver enhancing).

High Resolution A crisp image of separated protein bands is produced on the membrane.

Easy to Use The single solution is used straight from the bottle.

Economical PROTOGOLDTM may be re-used on successive blots until the gold is exhausted.

Quantitative Determination Proteins are detectable from solution below 1ng/ml.

Cannot Overstain The membrane may be left in solution. No destaining is necessary.

Long Shelf Life PROTOGOLDTM is stable at 2-8°C for until its expiry date. This product cannot be frozen.

Gold Stains


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Method of UseThe application of PROTOGOLDTM to proteins blotted on a membrane is very straightforward.

a) Incubate the blotted membrane with 0.3% Tween20 detergent to block unoccupied sites and to eliminate background.

b) Wash in deionised water to remove buffer salts

c) Incubate the membrane with PROTOGOLDTM in a tray with continuous gentle agitation. After 2 to 3 minutes, proteins begin to appear stained light red. After 10 minutes the red colour deepens on the strongest bands. Optimal staining occurs after 2 hours. The membrane may be incubated indefi nitely without overstaining. There is no need to de-stain.

If necessary the membrane can then be silver enhanced to give a further 10 to 100 times signal intensity with the BBI BL Silver Enhancing Kit.

d) Wash the membrane in water and dry.

Quantitation of Proteins from SolutionIndividual proteins in solution can easily be quantifi ed by direct dot blotting onto nitrocellulose and staining with PROTOGOLDTM. The resulting stain is compared with standards or a titrated series of protein blots stained simultaneously. Less than 1ng/ml of protein can be detected by this method.

Further Technical HelpFurther advice on using PROTOGOLDTM in the technical instruction booklet. PROTOGOLD® is a regestered trademark of BBInternational Ltd

Tween20® is a regestered trademark of Atlas Chemical Industries

Gold Stains

Product Unit Size Product Code

PROTOGOLD® Kit 500ml PRO500 £107.00

Tween20® detergent 10ml T20 £9.00

Price

077.000£1

9.000£99


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The BBI Silver Enhancing kits provide simple to use and sensitive systems for the amplifi cation of immunogold labels in light microscopy, electron microscopy and blotting applications. Enhancement occurs during the reduction of silver from one solution (the Enhancer) by another (the Initiator) in the presence of gold particles. This reduction causes silver to build up on the surface of the gold particles that are attached to antibodies, proteins or other macromolecules on the target specimen. Enhancement is rapid but easily controllable within a comfortable time span (minutes). Amplifi cation of the gold signal by 10 to 100 times is readily achieved. The reaction is insensitive to light, is simply stopped by washing in water, and needs no fi xing.

BBI provide two silver enhancing kits for different applications:

• LM/EM Silver Enhancing Kit - for light and electron microscopy

• Blotting Silver Enhancing Kit - for blotting applications

• Light Microscopy

Specimens may be immunogold labelled sections of tissue, whole cells, smears, chromosomes, etc mounted on glass slides, cell monolayers or tissue slices in culture. Small gold particles are not visible in the LM because of the limit of resolution (>200nm) of the microscope. After the immunogold labelling the specimen is washed in water and simply enhanced in a single step with the solutions provided. The fi nal result is an intense brown/black stain at the site of the gold label.

5nm immunogold labels give excellent results with silver enhancement, 2 nm gold labels being of particular value when penetration of cells is required. Adequate amplifi cation is usually obtained within a period of 5/10 minutes and may be monitored on the microscope during the reaction. The enhancement is performed by mixing together one drop of each solution and applying to the slide. The reaction can be performed with a cover

slip in place to allow high magnifi cation viewing, even with oil immersion. The reaction is stopped by simply washing in water. It may be continued if necessary with fresh solutions.

The LM/EM Silver Enhancing Kit is suffi cient for at least 300 incubations on glass slides. After washing, the slides may be counterstained and mounted as normal. Because of the discrete nature of the growth of silver on the gold particles, no diffusion of the signal occurs. The silver stain produces a permanent, non-fading label of sharp resolution and high contrast in bright fi eld viewing.

BIOMOUNT may be used to avoid leaching of the silver signal that may occur with other types of mountant.

Epipolarised LightIn conjunction with epipolarised light the gold/silver enhancement also gives a brilliant image of the signal against a dark background in the light microscope. In this arrangement, the epipolarised light source (usually UV) is refl ected by the perfectly spherical silver ‘mirrors’ formed by enhancement of the gold particles. Sensitivity is increased by 10 times in comparison with normal bright fi eld viewing. This means that lower levels of gold are needed for visualisation, thus producing a much improved peak to background signal. Most modern microscopes can easily and inexpensively be equipped with an epipolarising light attachment by the manufacturer.

Electron MicroscopyAny gold label on an electron microscope specimen may also be effectively enhanced with the LM/EM Silver Enhancing Kit. Silver enhancing for electron microscopy is especially useful where a low labelling intensity, due to very low levels of tissue antigen, requires that 1nm or 5nm gold conjugates must be used. With these small particles the best possible labelling intensity is achieved but the particles may not be so easily visualised, especially in electron microscopes of limited magnifying power. By fl oating the EM grid carrying the ultrathin specimen on a drop of enhancing solutions for a few minutes the particles become large enough to see at much lower magnifi cations. It is quite common to observe these particles, which can be grown to 50nm or more, at only 10,000 times magnifi cation or lower. The immunolabelling can therefore be observed within a

Silver Enhancing Products


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much wider specimen area than at high magnifi cation. The gold particles grow in a perfectly spherical manner but when adjacent particles touch they fuse to form elliptical shapes. The technique is so simple that specimens may be enhanced repeatedly and observed in the microscope until the required particle size is achieved.

Scanning Electron MicroscopyFor SEM studies any gold particle can be used in the immunogold reaction and these particles grown to a size (e.g. 50nm) which is convenient to view by scanning using backscattered electrons or X-ray mapping. The use of small gold particles, e.g. 5nm, with subsequent silver enhancing is a preferred approach to labelling SEM specimens compared to the use of larger gold particles for direct viewing because of the higher labelling intensity.

Blotting ApplicationsThe Blotting Silver Enhancing kit is formulated to produce an intense, sharp, black signal from gold labels on specimens such as proteins immobilised on

membranes. It is therefore ideal for enhancing immunogold or total gold stains from Western, Southern, Northern, dot blots, using the PROTOGOLD® and GENOGOLDTM products and the blotting grade immunogold reagents. With this system, immunogold/silver staining provides a safe, sensitive, rapid and economical alternative to radiolabelling procedures.

The Blotting Silver Enhancing Kit is provided as two separate solutions. Equal volumes (e.g. 10ml) of each solution are mixed before use. After gold staining, the membrane is washed in water and then simply immersed in the mixed solutions for the appropriate time. The reaction is stopped by washing in water.

PROTOGOLD®, is a registered trademark of BBInternational Ltd (BBI)

Test StripsThese strips allow the sensitivity and stability of the kit to be monitored over a period of time. They may also be used in parallel with each enhancing reaction. Packs of 10 Test Strips are available separately.

Silver Enhancing Products

Product Unit Size

LM/EM Silver Enhancing Kit 2 x 15ml >300 SEKL15 £78.00 BL Silver Enhancing Kit 2 x 250ml 20 to 30 blots SEKB250 £107.00 Test Strips Pack of 10 10 SETS10 £20.00

No. of Reactions Product Code

SEKKB2500

SETSS10

SEKKL15

Further technical and procedural help on LM/EM silver kits and BL silver kits, please contact our technical

support team on +44 (0) 29 2074 7232 or e-mail [email protected].

££78.000

££1077.000

££20.000

Price


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TUNICRYLTM is a unique acryllic resin whuch has been developed for universal use in both light and electron microscopy1. It has the following applications:

Light Microscopy Electron MicroscopyHistology UltrastructureHistochemistry CytochemistryImmunocytochemistry ImmunocytochemistryImmunolabelling ImmunolabellingIn Situ Hybridisation In Situ Hybridisation

UNICRYLTM provides optimum sectioning, labelling and staining qualities for studies in animal, plant and microbiological tissues

UNICRYLTM is uniqueThe advantages of UNICRYLTM for staining and labelling lie in both its preservation of tissue structures and its sectioning characteristics such that proteins, nucleic acids and macromolecules are revealed at the surface of the sections for subsequent incubations. The resin preserves these structures without chemically interacting or cross linking with them. UNICRYLTM is largely hydrophilic, allowing good access to polar (aqueous) solutions and exhibiting a low background staining or labelling from hydrophobic materials. It also minimises the denaturation of proteins, allowing true antigenic properties to be maintained. Normal counterstaining properties for both EM and LM studies are excellent due to the hydrophilicity of the resin and its homogeneous structure1,2,3,4,5.

UNICRYLTM is easy to useThe resin is provided as a single solution and is used in a similar way to other acrylic resins for embedding tissues. Because it is a single solution no mixing is required. The resin has a long shelf life if stored in the cold. It is miscible with alcohols and has a low viscosity even down to –50°C. UNICRYLTM can be polymerised by heat or by UV irradiation at lower temperatures. Tissue pieces are processed in single capped 1ml eppendorf tubes, BEEM capsules or gelatin capsules by simple pipetting of solutions into the tubes. Cells in culture may be processed in situ in vials enclosed during polymerisation.

StorageUNICRYLTM is stable at -20°C until its expiry date.

Each UNICRYLTM kit comprises 250ml of resin, embedding vials, plastic gloves and a disposable bottle. A detailed instruction booklet explaining methods for specimen preparation and polymerisation at high and low temperatures is provided with each UNICRYLTM kit.

The UNICRYLTM STAINING KIT is stable at 2-8°C, until its expiry date.

SafetyUNICRYLTM resin contains methacrylates, styrene and benzoyl peroxide. Please read the UNICRYLTM MSDS for safe handling and disposal information.

AcknowledgementsUNICRYLTM resin was originally developed by the research group of Dr C Scala at the Institute of Clinical Electron Microscopy, University of Bologna, Italy. In the papers of Scala et all, the UNICRYLTM resin is referred to as Bioacryl, its original name. The staining protocols follow modifi cations of the method of Scala et al.

Ordering Information

UNICRYLTM


UNICRYL® Resin Kit 250ml BA250 £146.006.006 0046£11

Price


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BIOBONDTM Tissue Section AdehsiveFor light microscopy and electron microscopy the choice of specimen preparation is critical for the preservation of antigens in the sample. Of greatest importance in the preparation schedules are the specimen fi xation and embedding. The protocol must satisfy the requirements for preservation of structural integrity and antigenicity.

Having prepared the tissue specimen for immunolabelling it is then imperative to perform the incubations with a protocol designed to maximise the specifi c signal and minimise the background. Some incubation conditions may cause tissue sections to be removed from the glass slide. Typical tissue section adhesives such as poly-L-lysine, Elmer’s glue, chrome alum, etc are not suitable for use with immunogold labelling because of the increased background caused by attraction of gold particles to the adhesive on the slide.

In addition the surface of glass slides is uneven and is activated by the silicon tetrahedral structure. This provides active sites for absorption of proteins or reactions with chemicals and reagents. It is therefore important to minimise this possibility by coating the surface with a material that is of low reactivity towards reagents.

BBI now offers a special slide coating solution, BIOBONDTM, which produces a very strong adhesion between the glass and the tissue section for subsequent incubations. BIOBONDTM coats the slide with a protective layer to minimise interaction of charged glass surface with reagents. This is also of particular importance for reproducibility of results because of the variations that occur between glass slides obtained from different sources and in different countries. It is particularly effective for use with severe incubating conditions such as those used in in situ hybridisation. BIOBONDTM is suitable for all kinds of tissue specimens including paraffi n wax or resin sections, cell smears, cytospins or cryostat sections. BIOBONDTM is supplied in 20m1 unit volumes, suffi cient to coat at least 1000 slides.

BIOMOUNTTM

Tissue Section Mounting MediumBIOMOUNTTM reduces fading of immunogold/silver signals in sections on glass slides. It is suitable for both resin and wax embedded tissue sections.

Some mounting media oxidise rapidly on exposure to air, forming carboxyl groups. This may be especially so when sections have been cleared in a solution containing aldehyde groups. It is sometimes observed that the immunogold / silver stain fades after a few weeks, or even in a shorter time, from sections that have been mounted with these media under cover slips. The silver is still present, but has formed translucent silver carboxylate salts. Visibility can be retrieved by removing the cover slip and washing in xylene (xylol) and then immersing the slide in photographic developer, but this is a tedious procedure. BIOMOUNTTM prevents this fading because of its low oxidising properties.

BIOMOUNTTM is miscible with xylene and may be applied to sections on slides in the normal manner following dehydration and immersion in xylene. Labelling will retain its intensity and contrast following the use of BIOMOUNTTM. Sections that have been counterstained with normal stains or with the UNICRYLTM STAINING KIT will also retain their colour intensity.

Ancilliary Products


BIOBONDTM 20ml BB20 £36.00Tissue Adhesive

BIOMOUNTTM Tissue 100ml BM100 £25.00Mounting Medium

00000036..0££33

00025..0££22

Price


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Blocking ReagentsNon-specifi c labelling can occur on specimens during immunolabelling procedures. The source of this background labelling must be determined by the careful and systematic use of controls and eliminated for a proper analysis of the specimen. Background labelling can occur from a number of sources, either in the specimen or in the incubating solutions. In either case the background can be substantially reduced by the careful use of blocking reagents.

• Background labelling from the specimen• Hydrophobic attraction of proteins or gold

particles • Charge attraction from tissue components to

antibodies or gold particles • Fixing of antibodies through excess aldehyde

groups in tissue • Receptors in tissues for Fc components of

antibodies • Excess sulphur in tissue or support • Excess lysine in tissue adhesive (for LM

sections) • Background labelling from incubating

solutions• Hydrophobic proteins (e.g. antibodies) • Charged proteins (e.g. pH, ionic

concentration) • Charged gold particles • Gelatin in buffer

Most of these sources of background can easily be overcome by the addition of appropriate blocking reagents to the buffers used for incubating or by a separate blocking step before the primary antibody. Full details of the correct use of controls to identify the background source and the subsequent use of blocking reagents is given in the Conjugate Technical Information section.

Ancilliary ProductsProduct Unit Size Product

Code

Tween 20® 10ml T20 £9.00 BSA (fatty acid free) 10g BSA10G £39.00 Gelatin (fi sh 45%) 10ml GEL10 £20.00 Tween 20® is a registered trade mark of Atlas Chemical Industries

Price

£99.000

£339.000

£220.000


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Ordering InformationBBI is a global supplier and has 25 years’ experience in the manufacture of superior quality gold nanoparticles and secondary gold probes for the Life Sciences and nanotechnology fi eld. Our products are guaranteed for a high level of stability and consistency and our customers are supported by a team of technical support specialists and distributors in over 200 countries.

Browse our products and services online or contact one of the friendly BBI team to fi nd out how we can help you.

Ordering Directly From BBIYou can place an order quickly and securely by credit/debit card using our online shopping cart.

Alternatively if you do not have the ability to use a credit or debit card you may order direct from us. BBI accepts institutional purchase order from all over the world. You can place your order by contacting us:

By Telephone +44 (0) 29 20 74 7232

Fax your order to: +44 (0) 29 20 74 7242

E-mail your order to: [email protected]

Online Ordering InstructionsTo ensure your privacy and safety when registering and ordering online all communications between your web browser and our server is encrypted using a secure socket layer (SSL) protocol.

To order securely online fi rstly select the desired currency at the top of the page (Prices listed are in GBP and in US dollars for international customers). Then choose the products of interest in the size, volume and quantity you require. Once selected, add your products to the shopping cart using the “Buy” button.

When you are done shopping, navigate to the checkout page using the top right menu. When placing your fi rst order you will be asked to register for an online account. Registering for an account also gives you access to special offers, exclusive promotions, newsletters and new product information.

Shipping, Return & Replacement PolicyPlease review our shipping policy page on www.buybbi.com for more information.

How to Order


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AustraliaTaylor Biomedical PTY Ltd5/ 31 Howe StreetVictoriaPhone: +61280844762E-Mail: offi [email protected]

AustriaChristine Groepl ElectronenmikroskopieFrauenhofnerstrasse 40A-3430 TullnAustriaPhone: + 43 22 726 3177Fax: + 43 22 726 31774E-mail: [email protected]

BelgiumTEBU-BIO NVAlexander Franckstraat 196B-2530 BoechoutPhone: + 03 454 00 66Fax:+ 03 454 18 88E-mail : [email protected]: www.tebu-bio.com

CanadaCEDARLANE4410 Paletta CourtBurlingtonON L7L5R2CanadaPhone: 1-800-268-5058Fax: 1-289-288-0020E-mail: [email protected]: www.cedarlanelabs.com

ChinaUnisize Technology (Changzhou) Co., Ltd. No.801, Middle Road Chang Wu, Wujin District,Changzhou, Jiangsu ProvincePhone/Fax: +86-519-81195258Email: [email protected]: www.unisizetech.com

Pin-An-Nuo Technology (Beijing) Co., Ltd.Department shuiqingmuhua, zhongguancun, Haidian District,Beijing, 100190Manager: Xinyue WenQQ: 497465865Email: [email protected] Beijing LeBo Biotech CompanyRoom 912,9f, Tongyuan Tower, No.95, Qinghe 3th Street,Haidian District, BeijingPhone: (86)-10-59564854 (86)-10-59564854Fax: (86)10-62955993E-mail: [email protected]: www.lab-bio.com

Czech Republic & Eastern EuropeBARIA s.r.o.Jižní 393252 44 PsáryCzech RepublicPhone/Fax: 00420 323 550 123Email: [email protected]: www.baria.cz

Denmark, Finland and NorwayCytotech - Nota Bene Scientifi c ApSNordre Strandvej 119FDK-3150HellebaekDenmarkPhone: +45 70 23 20 60Fax: +45 70 23 20 80E-mail: [email protected]: www.nbs.dk

BBI Distributors


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FranceTEBU-BIO SA39, Rue de HoudanF-78612 Le Perray en YvelinesCedexPhone: +01 30 46 39 00Fax: +01 30 46 39 11E-mail: [email protected]: www.tebu-bio.com

GermanyPLANO W PLANNET GmbHErnst-Befort-Strasse 1235578, WetzlarGermanyPhone: +49 6441 97 650Fax: +49 6441 976 565Email: [email protected]: www.plano-em.com POLYSCIENCES EUROPE GmbHHandelstr 3D 69214, EppleheimGermanyPhone: +49 6221 765 767Fax: +49 6221 764 620E-mail: [email protected]: www.polysciences.com

Hong KongADVANCED TECH. & IND. CO., LTD.UNIT B, 1/F, Cheong Shing Ind. Bldg.,17 Walnut St., Tai Kok Tsui, Kln, HONG KONGPhone: 23902293 Fax: 27898314Email: [email protected]

IsraelBIO-NET26 Rachel ImenouJerusalem 93228IsraelPhone: +972 2 567 1553Fax: +972 2 563 1963E-mail [email protected]

ItalyProdotti Gianni S.p.A.Via Quintiliano, 30 - 20138Milano ItalyPhone: +39 02 509 7 509Fax: +39 02 509 7 276E-mail: [email protected] Web: www.ricerca.it

JapanFUNAKOSHI Co. Ltd9-7 Hongo 2-chomeBunkyo-kuTokyo 113-0033JapanPhone: +81 3 5684 1620Fax: +81 3 5684 1775E-mail: [email protected]: www.funakoshi.co.jp KoreaWOOMYOUNG INC5F, KT Building, 734 Suseo-DongKangnam-Gu, Seoul 135-886KoreaPhone: +82-2-3412-6999Fax: +82-2-417-2863E-mail: [email protected] Web: www.woomyoung.co.kr

MalaysiaSynergy Scientifi c Sdn Bhd29-2, Plaza Danau 2, Jalan 109F, Taman Danau Desa, 58100Kuala Lumpur, MalaysiaPhone: +603 79832632Fax: +603 79832635E-mail: alycia@synergyscientifi c.com.my

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NetherlandsSanverTECH bvBevelandseweg 28NL-1703-AZHeerhugowaardThe NetherlandsPhone: +31 72 572 2100Fax: +31 72 572 2133E-mail: [email protected]: www.tebu-bio.com

New ZealandTaylor Biomedical PTY Ltd5/ 31 Howe StreetVictoriaPhone: +61280844762E-Mail: offi [email protected]

Singapore & South East AsiaSynergy Scientifi c Sdn Bhd29-2, Plaza Danau 2, Jalan 109F, Taman Danau Desa, 58100Kuala LumpurMalaysiaPhone: +603 79832632Fax: +603 79832635E-mail: alycia@synergyscientifi c.com.my

SpainAbyntek Biopharma,S.L.Parque Tecnológico de BizkaiaEdifi cio 50448160 Derio - BizkaiaPhone: (+34) 94 404 8080Fax: (+34) 94 404 8081E-Mail: [email protected]: www.abyntek.com

VITRO S.A.Apartado No. 1286 F.D.28080 MadridSpainPhone: +34 91 535 39 60Fax: +34 91 535 27 80E-mail: [email protected]: www.vitrosp.com

SwedenANALYTICAL STANDARDS ABMetallvagen 5S0-43533, MolnlyckePhone: +46 31 88 08 10Fax: +46 31 88 08 86E-mail: [email protected]: www.nordlab.se

SwitzerlandCHEME BRUNSCHWIG AGBelchenstrasse 12Postfach, CH 4009BaselSwitzerlandPhone: +41 61 308 91 15Fax: +41 61 308 91 19E-mail: [email protected]: www.brunschwig-ch.com

Thailand & South East AsiaSynergy Scientifi c Sdn Bhd29-2, Plaza Danau 2, Jalan 109F, Taman Danau Desa, 58100Kuala LumpurMalaysiaPhone: +603 79832632Fax: +603 79832635E-mail: alycia@synergyscientifi c.com.my

TaiwanHygene Biotech Company Ltd1F., No.10,Lane 4, Sanfu St.,Wenshan District, Taipei City 116, TaiwanPhone: 886-2-2933-6382 Fax: 886-2-8663-1471E-mail: [email protected] Web: www.hygene.com.tw

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LEVEL BIOTECHNOLOGY Inc9F-3 no 21Lane 169 Kong Kin StreetHis-Chih 221, Taipei HsienTaiwanPhone: +886 2 2695 1252Fax: +886 2 2695 7393Web: www.level.com.tw

United Kingdom2B Scientifi c LtdCherwell Innovation Centre77 Heyford ParkUpper HeyfordOX25 5HDPhone: +44(0) 1869 238033Fax: +44(0) 1869 238034Email: farjada@2BScientifi c.com Web: www.2BScientifi c.com

AGAR66A Cambridge RoadStansteadEssexPhone: + 44 (0) 1279 813 519Fax: + 44 (0)1279 815 106E-mail: websales@agarscientifi c.comWeb: www.agarscientifi c.com

USATed Pella, Inc.4595 Mountain Lakes BoulevardReddingCA 96003USAPhone: + 530 243 2200Fax: + 530 243 3761E-mail: [email protected]: www.tedpella.com

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