ELECTROPHORESIS

Presented by:Utkarsh AlokM.Pharm 1st semDept. Of PharmacologyRIPS

Under the guidance of:Sasmita Kumari AcharjyaAsst.ProfessorDept. of Pharmaceutical analysisRIPS

CONTENTSINTRODUCTIONTHEORYTYPES OF ELECTROPHORESISMOVING BOUNDARY ELECTROPHORESISZONE ELECTROPHORESISPRINCIPLE AND TYPES OF ZONE ELCTROPHORESISGEL ELECTROPHORESIS PRINCIPLE OF GEL ELECTROPHORESISAGAROSE GEL PREPRATION AND INSTRUMENTATIONSAMPLE PREPRATIONGEL LOADING AND MICROPIPETTING TECHNIQUEGEL STAININGPOLY ACRYLAMIDE GEL ELECTROPHORESIS (PAGE)PREPRATION OF PAG AND ITS COMPONENTSCOMPARISION OF FLATBED SYSTEM AND VERTICAL GEL SYSTEMISO-ELECTRIC FOCUSSINGSDS-PAGE

2-D ELECTROPHORESISCAPILLARY ELECTROPHORESIS-PRINCIPLE AND COMPONENTSCOMPARISION BETWEEN CAPILLARY ELECTROPHORESIS AND HPLCCOMPARISION BETWEEN CLASSICAL GEL AND CAPILLARY ELECTROPHORESISREFRENCES

ELECTROPHORESIS

Electrophoresis is also called Cataphoresis.

Electrophoresis is a separation technique in which ions in colloidal solutions are separated based upon their differences in size and charge when a high voltage is applied to the solution[1]

Positive ions migrate to the negative electrode (cathode), and negative ions migrate towards the positive electrode (anode).

Was developed into workable system by Arne Tiselius of Sweden in 1930. He won Nobel prize for the work in 1948.

Introduction:

Theory of electrophoresis

Mobility- rate of movement ,denoted by “μ”.Particle to migrate in electric field requires a net electrostatic charge.Consider a particle that is placed in a container of liquid, saturated with the buffer and has a potential applied to it. The force “F” is equal to: F= QE Where, Q= charge of the particle E= field strength

As particle moves in the buffer it meets retardation force caused by the viscosity of the solvent which can be expressed as: Fs= 6πrημ Where, fs = viscous retardation force r = radius of particle in cm η = viscosity of mediun in poise μ = electrophoretic velocity in cm/sec

Now when both the forces are equal i.e Fs = QE = 6πrημ

Hence mobility of particle can be defined as: μ = υ/E where, μ = mobility in cm2volt-

1sec-1

E = volt cm-1

After substitution, μ = Q/6πrη

Factor affecting the mobility[1]

Heat productionSmilingElectro-osmosis correction

Moving boundary electrophoresis :

Density gradient electrophoresis

Iso-tachophoresis

Zone electrophoresis:

Paper electrophoresis

Gel electrophoresis

Capillary zone electrophoresis

Immuno- electrophoresis

Iso-electric focusing electrophoresis

Types of electrophoresis

Separation occurs due to difference in mobility of molecules. Mobility is proportional to m/e ratio.The position of moving ions, which forms a boundary, which is detected by measuring the changes in refractive index throughout solution.The concentration gradients which are formed during electrophoresis are usually detected by optical method.

Moving boundary electrophoresis[2]

This method allows the charged species to migrate in a free moving solution in absence of a supporting medium.Samples are fractioned in a U shaped tube that has been filled with unstabilized buffer.An electrical field is applied by means of electrodes at the ends of the tube.

Fig1: illustration of moving boundary electrophoresis

Advantages:1.Application to a wide variety of high molecular weight substances.

Disadvantages [3]

1.Mixing of separated compound as a consequence of thermal and density gradient as well as mechanical vibration.2.Thermal vibration and mechanical vibration controlling is difficult and expensive.3.Detection of fraction separated with optical system also adds to the expenses.

ADVANTAGE AND DISADVANTAGES OF MOVING BOUNDARY ELECTROPHORESIS

ZONE ELECTROPHORESIS

Any electrophoretic technique in which components are separated into zones or bands in a buffer and stabilized in solid, porous, or any other support medium e.g.: paper strip, agar gel or poly-acrylamide gel.

Separates macromolecular colloids e.g.. proteins in serum, urine, Cerebrospinal fluids (CSF), erythrocytes; nucleic acids.

Types of zone electrophoresis are:-

1. Paper electrophoresis

2. Gel electrophoresis

3. Capillary zone electrophoresis

4. Immuno- electrophoresis

ADVANTAGES AND DISADVANTAGES OF ZONE ELECTROPHORESIS

ADVANTAGES:1. Useful in biochemical investigation.2. Very small quantity of samples can be analyzed.3. Useful to study both simple and complex mixtures equally.4. Equipment cost is low and maintenance is easy.5. Detection and visualization with various reagents and dyes

are possible6. Permits 2-D electrophoresis for higher resolution.7. Quantification by densitometry and auto-radiography can

be done

DISADVANTAGE:1. Unsuitable for accurate mobility and iso-electric point

determination.2. Complications such as capillary flow, electro osmosis,

adsorption and molecular sieving are introduced.

GEL ELECTROPHORESIS[4]

Gel electrophoresis is a widely used technique for the analysis of nucleic acids and proteins. Gel electrophoresis is routinely used for the preparation and analysis of DNA.Gel electrophoresis is a procedure that separates molecules on the basis of their rate of movement through a gel under the influence of an electrical field.

Types of gel electrophoresis:-

1. One-dimensional - Agarose gel electrophoresis, Poly-acrylamide gel electrophoresis (native or SDS-PAGE) and Iso-electricfoccusing (IEF) or

2. Two dimensional - 2D-PAGE.

3. Capillary electrophoresis

+-

Power

DNA

PRINCIPLE OF GEL ELECTROPHORESIS

DNA is negatively charged When placed in an electric field, DNA will migrate towards the positive pole (anode).An agarose gel is used to slow the movement of DNA and separate by size.

Fig 2 :Scanning Electron Micrograph of Agarose Gel (1×1 µm)

• Polymerized agarose is porous, allowing for the movement of DNA

+-

Power

DNA

How fast will the DNA migrate?

• Strength of the electrical field, buffer, density of agarose gel

• Size of the DNA• Small DNA move faster than large DNA• Gel electrophoresis separates DNA according to size

smalllarge

Within an agarose gel, linear DNA migrate inversely proportional to the log10 of their molecular weight.

AGAROSE [5]

•Agarose is a linear polymer extracted from seaweed.

• 0.7% - for large DNA (5-10 kb)

• 2%- for small DNA (0.2-1kb)

• Recommended concentration is 1%

•Agarose was first used in biology when Robert Koch used it as a culture medium for Tuberculosis bacteria in 1882

Fig 3:- Agarose used for electrophoresis

Making an Agarose Gel

An agarose gel is prepared by combining agarose powder and a buffer solution.

Agarose

Buffer

Flask for boiling

Buffer used- Tris-borate buffer (pH 8.0) that contains EDTA (TBE); EDTA is a chelating agent that binds divalent cations such as Mg++ that many nucleasesrequire for their activity; EDTA thus protects the DNA from enzymatic degradation[6]

Fig: 4

Casting trayGel combs

Power supply

Gel tank Cover

Electrical leads

Electrophoresis Equipment

Fig 5: Electrophoresis Equipment

Gel casting tray & combs

Fig :6

Small 8x10 cm gels (minigels) are very popular.The volume of agarose required for a minigel is around 30–50 mL, for a larger gel it may be 250 mL.

Seal the edges of the casting tray and put in the combs. Place the casting tray on a level surface. None of the gel combs should be touching the surface of the casting tray.

Preparing the Casting Tray

Fig: 7

Fig 8:Agarose Fig: 9 Buffer Solution

Combine the agarose powder and buffer solution. Use a flask that is several times larger than the volume of buffer.

Fig 10: Agarose is insoluble at room temperature.

Gently swirl the solution periodically when heating to allow all the grains of agarose to dissolve. Be careful when boiling - the agarose solution may become superheated and may boil violently if it has been heated too long in a microwave oven.

Melting the Agarose

Fig 11: The agarose solution is boiled until clear.

Allow the agarose solution to cool slightly (~60ºC) and then carefully pour the melted agarose solution into the casting tray. Avoid air bubbles.

Pouring the gel

Fig :12

Each of the gel combs should be submerged in the melted agarose solution.

Fig : 13

When cooled, the agarose polymerizes, forming a flexible gel. It should appear lighter in color when completely cooled (30-45 minutes). Carefully remove the combs and tape.

Fig : 14

Place the gel in the electrophoresis chamber.

Fig: 15

buffer

Add enough electrophoresis buffer to cover the gel to a depth of at least 1 mm. Make sure each well is filled with buffer.

Cathode(negative)

Anode(positive)

wells

DNA

Fig: 16

6X Loading Buffer: Bromophenol Blue (for color) Glycerol (for weight)

Sample Preparation

Mix the samples of DNA with the 6X sample loading buffer (w/ tracking dye). This allows the samples to be seen when loading onto the gel, and increases the density of the samples, causing them to sink into the gel wells.

Fig 17: prepared DNA sample

Loading the Gel

Carefully place the pipette tip over a well and gently expel the sample. The sample should sink into the well. Be careful not to puncture the gel with the pipette tip.

Fig 18

Micropipette tip should be ABOVE the well NOT IN IT!!!!

MICROPIPETTING TECHNIQUE

Fig 19

Micropipette tip punched

right through the gel

See dye under the wells

Improper technique

Fig 20

Correct technique

Fig 21

Place the cover on the electrophoresis chamber, connecting the electrical leads. Connect the electrical leads to the power supply. Be sure the leads are attached correctly - DNA migrates toward the anode (red). When the power is turned on, bubbles should form on the electrodes in the electrophoresis chamber.The electrophoresis is run by 70-100 V/20-80 mA for about an hour or at 20 to 30 V overnight [6]

RUNNING THE GEL

Fig 22 Fig 23

wells

Cathode(-)

Anode(+)

Gel

After the current is applied, make sure the Gel is running in the correct direction. Bromophenol blue will run in the same direction as the DNA.

DNA(-)

Fig 24

100 200 300

1,650

1,000

500

850

650

400

12,000 bp

5,000

2,000

DNA LADDER STANDARD

Inclusion of a DNA ladder (DNAs of know sizes) on the gel makes it easy to determine the sizes of unknown DNAs.

-

+

DNAmigration

bromophenol blue

STAINING THE GEL

***CAUTION! Ethidium bromide is a powerful mutagen and is moderately toxic [7]

• Ethidium bromide binds to DNA and fluoresces under UV light, allowing the visualization of DNA on a Gel.

• Ethidium bromide can be added to the gel and/or running buffer before the gel is run or the gel can be stained after it has run.

SAFER ALTERNATIVES TO ETHIDIUM BROMIDE

Methylene Blue Carolina BLU Stain EVA green [8]

AdvantagesInexpensiveLess toxicNo UV light requiredNo hazardous waste disposal

DisadvantagesLess sensitiveMore DNA needed on gelLonger staining/destaining time

stains Compounds

Amido black 10 B Proteins

Coomassie blue Proteins

Sudan black Lipid & lipoproteins

Ninhydrine Amino acid

Other stains which are used [1]

STAINING THE GEL

• Place the gel in the staining tray containing warm diluted stain.• Allow the gel to stain for 25-30 minutes.• To remove excess stain, allow the gel to destain in water.• Replace water several times for efficient destain.

Fig 27

Ethidium Bromide requires an ultraviolet light source to visualize

DNA bands under UV light in an ethidium-bromide-stained gel is easily visible if it contains about 20 ng of DNA.

Fig 28

POLYACRYLAMIDE GEL ELECTROPHORESIS (PAGE)

PAGE, is one of the most widely used electrophoresis techniques and separates proteins through a polyacrylamide gel matrix[4]

Two types of PAGE can be carried out 1.Native PAGE- in which electrophoresis is carried out under non-denaturing conditions and separation is based on the protein’s charge and hydrodynamic size or2. SDS PAGE - in which proteins are denatured prior to electrophoresis and separation is based on a protein’s mass or molecular weight. The chemical SDS (sodium dodecyl sulphate) is an anionic detergent which, in combination with DTT (dithiothreitol) or β-mercaptoethanol, breaks intramolecular bonds in the protein destroying any secondary, tertiary or quaternary structure. This leaves only the linear primary amino acid structure of the protein which will contain an overall negative charge proportional to its mass, therefore allowing the proteins to be separated solely on the basis of their molecular mass.

The gel typically consist of acrylamide, bisacrylamide, SDS, and a buffer with an adjusted pH. The solution may be degassed under a vacuum to prevent the formation of air bubbles during polymerization. A source of free radicals and a stabilizer such as ammonium persulfate and TEMED are added to initiate polymerization. The polymerization reaction results in a gel because of the added bisacrylamide, generally about 1 part in 35 relative to acrylamide, which can form cross-links between two polyacrylamide molecules. The ratio of acrylamide to bisacrylamide can be varied for special purposes. The acrylamide concentration of the gel can also be varied, generally in the range from 5% to 25%.Lower percentage gels are better for resolving very high molecular weight proteins, and viceversa.

PREPARING POLY-ACRYLAMIDE GELS[9]

COMPONENTS OF POLYACRYLAMIDE GEL ARE [9]:-

1.Chemical buffer -to stabilizes the pH value to the desired value-

Tris , Bis-Tris or imidazole.

2.Counterion -to balance the intrinsic charge of the buffer ion and

also affect the electric field strength during electrophoresis – glycine

and tricine.

3.Acrylamide.

4.Bisacrylamide - used as cross linking agent .

5.Sodium Dodecyl Sulfate - denature native proteins

6.Ammonium persulfate - as an initiator for gel formation.

7.TEMED (N, N, N', N'-tetramethylethylenediamine)- stabilizes

free radicals and improves polymerization.

CHEMICALS FOR PROCESSING AND VISUALIZATION Tracking dye:- Anionic dyes of a known electrophoresis mobility are

used like Bromophenol blue to follow colorless protein .

Being a highly mobile molecule it moves ahead of most proteins. As it

reaches the anodic end of the electrophoresis medium electrophoresis

is stopped.

Loading aids:- To ensure that the sample sinks to the bottom of the

gel  glycerol and sucrose are used to increase the density of the

sample.

Coomassie Brilliant Blue R-250 (CBB) - is an anionic dye.

Proteins in the gel are fixed by acetic acid and simultaneously stained.

The excess dye incorporated into the gel can be removed by

distaining with the same solution without the dye. The proteins are

detected as blue bands on a clear background

Fig 30 : vertical gel slab illustrating PAGE

ADVANTAGES OF PAGE ELECTROPHORESIS

1. Excellent separation on basis of size, shape, and charge

2. Pore size and the amount of cross linking can be controlled

3. Excellent resolution

4. Separation is rapid (30 min to a few hours)

5. Apparatus is relatively simple to operate

6. PAGE has a high loading capacity, up to 10 micrograms of

DNA can be loaded into a single well (1 cm x 1 mm) without

significant loss of resolution.

7. PAGE is an ideal gel system from which to isolate DNA

fragments for subcloning and other molecular biological

techniques.

As any other methods, PAGE also has disadvantages:

1. The mobility of the fragments can be affected by base composition making accurate sizing of bands a problem.

2. Polyacrylamide quenches fluorescence, making bands containing less than 25 ng difficult to visualize with ethidium bromide staining.

3. Quantification difficult by itself - Increased when combined with procedures using radiolabeled or photometric markers

4. Acrylamide is a neurotoxin

Flatbed Systems: Vertical Systems:

Gel thickness is limited, becausecooling is only possible from one side

Higher protein loading capacity,because thicker gels can be used,which are cooled from both sidesBlotting is easier because of highergel thickness

One gel per instrument is run Multiple gel runs possible

Very versatile for different methods,ideal for isoelectric focusing

Limited technical possibilities, notoptimal for isoelectric focusing

Thin layers can easily be used,easy sample application

The thinner the gel, the more complicated is sample application

Easy to handle and to clean,no glass plates necessary, thus idealfor routine applications

Many pieces to set up and to clean

COMPARISON OF FLATBED AND VERTICAL GEL SYSTEMS[4]

ISO ELECTRIC FOCUSSING[1] [4]

It is mainly used for a separation of electrolytes such as proteins.When electrophoresis is run in solution buffered at constant pH, proteins having net charge will migrate towards opposite electrode.The use of pH gradient across supporting medium causes each protein to migrate to an area of specific pH.A sharp well defined protein bands occur at the point where iso electric point equals to pH of gradientSeparation is carried out on gels on which a stable a pH gradient has been established.An ampholytic compound has a pH at which it is neutral.The pH gradient is achieved by impregnating the gel with polyamino-polycarboxylic acids.When subjected to electric field these migrate and come to rest in order of their pH.Thus each ampholyte migrates in applied field until it reaches a position on the plate where pH of medium is equal to iso electric point.

At this point ampholyte is in its zwitter ion form and is neutral.Thus it losses electrophoretic mobility and becomes focused in narrow zone at this point.

Fig 31: illustrating IEF

Advantages:1.Spreading of bands is minimized.2.Proteins that differ by little as 0.01pH can be adequately resolved

Disadvantages:1.As carrier ampholytes are used in high concentration, a high voltage power supply is necessary. As a result the electrophoretsis may be affected.

ADVANTAGES AND DISADVANTAGES OF IEF

SDS-PAGE (PolyAcrylamide Gel Electrophoresis)[9]

SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis, is a technique widely used in biochemistry, forensics, genetics and molecular biology:1.To separate proteins according to their electrophoretic mobility (a function of length of polypeptide chain or molecular weight). 2.To separate proteins according to their size, and no other physical feature.SDS (sodium dodecyl sulfate) is a detergent (soap) that candissolve hydrophobic molecules but also has a negative charge(sulfATE) attached to it. SDS (the detergent soap) breaks up hydrophobic areas and coats proteins with negative charges thus overwhelming positive charges in the protein. The detergent binds to hydrophobic regions in a constant ratio of about 1.4 g of SDS per gram of protein. Therefore, if a cell is incubated with SDS, the membranes will be dissolved, all the proteins will be solublised by the detergent and all the proteins will be covered with many negative charges

Fig 32 : illustrating the effect of SDS over protein moleculesfi

COMPONENTS OF SDS PAGE GEL[9]

Running Buffer: Tris/Glycine: Glycine(pKa=9.69) is a trailing ion (or slow ion). In other words it runs through the gel slower then the slowest protein at a pH above 8.0.

Stacking Gel: Is prepared w/Tris/HCL buffer pH 6.8, ~2pH units lower than running buffer. Large pore polyacrylamide used to align and create a thin starting zone of the protein of apx. 19µm on top of the resolving gel.

Resolving Gel: Small pore polyacrylamide gel (3 - 30% acrylamide monomer) typically made using a pH 8.8 Tris/HCl buffer. Resolves protein ~24 – 205 kDa

Fig: 32

TWO DIMENSIONAL ELECTROPHORESIS

Separation of hundreds of proteins based on:Isoelectric pointMolecular weight

Popular method for protein display and proteomics-one spot at a time

Permits simultaneous detection, display, purification, identification, quantification, pI, and MW.

Robust, reproducible, simple, cost effective, scalable

Provides differential quantification using Differential 2D Gel Electrophoresis (DIGE)

Fig33: The principle of the 2-D electrophoresis according to O’Farrell (1975).

PROCESSES INVOLVED IN 2D GEL ELECTROPHORESIS

Protein isolation and quantification

Isoelectric focusing (first dimension)

SDS-PAGE (second dimension)

Visualization of proteins spots with Dye

Identification of protein spots with Mass Spec

CAPILLARY ELECTROPHORESIS[1]

Also known as: High performance capillary electrophoresis Capillary zone electrophoresis Free solution capillary electrophoresisCapillary electrophoresis is a micro-electrophoretic system in which separation takes place in a 10-100μm internal diameter, fused quartz, hollow capillary tube from 30-100 cm long with each end immersed in a buffer.DC applied upto 300V/cm.

WHY CAPILLARY ELECTROPHORESIS?Reduces the problem resulting from heating effect- more surface area to volume ratio hence more heat dessipation.Reduce zone broadening.

Forces associated with the capillary electrophoresis areElectro-osmotic flow (EOF)Electro-phoretic separationUnder these influence all components in the sample travels in one direction towards cathode.The electro-osmotic flow (EOF) is caused by applying high-voltage to an electrolyte-filled capillary. This flow occurs when the buffer running through the silica capillary has a pH greater than 3 and the SiOH groups lose a proton to become SiO- ions.  The capillary wall then has a negative charge, which develops a double layer of cations attracted to it.  The inner cation layer is stationary, while the outer layer is free to move along the capillary.  The applied electric field causes the free cations to move toward the cathode creating a powerful bulk flow. in CE potential applied is greater then normal electrophoresis i.e 300V/cm this facilitates electro-phoretic separation.

PRINCIPLE OF CAPILLARY ELECTROPHORESIS

Fig 34: illustrating the electro-osmotic flow in CE

COMPONENTS OF CE

1. COLUMN2. SAMPLE3. BUFFERS4. MODIFIERS5. POWER SUPPLY6. DETECTORS

COLUMNS:- borosilicate or fused quartzDimensions : internal diameter 10-100μm length 30-100cm capillary wall 300-600 μmSAMPLE:- 5-50nL Added by placing one end of the capillary into sample

containers raised to a set height (10cm) for a measured time (30sec) and allowing a siphoning action takes place or place one end into the sample and apply a potential for a short time.

BUFFERS: 0.5mM concentration is common 10mM mannitol 0.05M pH 7 phosphate, borate etc

MODIFIERS:- used if EOF is not demanded. these alter the direction and rate of EOF reverse flow- cetyltrimethyammoniunbromide tetradecyltrimethylammonium bromide zero flow-s-benzylthiouronium chloride reduce flow- methanol increase flow- acetonitrile

POWER SUPPLY:- faster heat dessipation hence high potential of 300V/cm can be applied.

DETECTORS:- U-V-visible detectors, fluorescence detectors, mass spectrometry,radioisotopes, conductometry and amperometry etc

1. CE has a flat flow, compared to the pumped parabolic flow of the HPLC. The flat flow results in narrower peaks and better resolution. 2. CE has a greater peak capacity when compared to HPLC—CE uses millions of theoretical plates.3. HPLC is more thoroughly developed and has many mobile and stationary phases that can be implemented.4. HPLC has more complex instrumentation, while CE is simpler for the operator.5. HPLC has such a wide variety of column lengths and packing, whereas CE is limited to thin capillaries.6. CE require less sample volume then HPLC.

Capillary Electrophoresis versus High Performance Liquid Chromatography (HPLC) [10]  

CE HPLC

Length of column 30-100cm 3-25 cm,25-50cm(microbore)

Internal diameter 10-100μm 4-6 mm,1-2 mm(microbore

Sample volume 5-50 nL 20μL

COMPARISION OF CLASSICAL GEL AND CE

Classical gel electrophoresis Capillary electrophoresis

Gels – polyacrylamide or agarose,Slabs length and width 5-25 cmColumns length- 7-10cm,internal diameter- 5mm

Fused quartz capillaryLength- 30-100cm, internal diameter-10-100μm

Electrophoretic separation Electrophoretic and electro-osmotic

Applied field100-2kV Applied field 10-50 kV

Heat dessipation is slow from column and quicker in slabs

Heat dessipation is rapid

0.05-0.5M electrolyte for conductivity and stabilitySample volume 1-50 μLDifferent sample can be analysed

Same as classical gel

Sample volume 1-50nLOnly one sample analysed at a time

Chromogenic agent or staining agent for detection of solute

HPLC detectors ,U-V absorbance and fluorescence are commonly used

Slow, limited resolution and time consuming.

High resolution, efficiency and sensitivity, is modrately fast

ADVANTAGES AND DISADVANTAGES OF CE

AdvantagesOffers new selectivity, an alternative to HPLC Easy and predictable selectivity High separation efficiency (105 to 106 theoretical plates) Small sample sizes (1-10 ul) Fast separations (1 to 45 min) Can be automated

DisadvantagesCannot do preparative scale separationsReproducibility problems

REFRENCES

1. Milons

2. Elctrophoresis in practice, fourth edition Reiver Westermeir

3. Instrumental method of chemical analysis. By B.K.Sharma, page

no- 269

4. Randox Research & Development catalogue 2009/10

5. HHMI undergraduate research studio – Freshman, Biology

section, Fall 2007 – Agarose Gel Electrophoresis.

6. www.wikipedia.org/wiki/Gel_Electrophoresis.

7. www.wikipedia.org/wiki/ethidium_bromide

8. Biotech.about.com/../DNA strain.htm

9. www.wikipedia.org/wiki/SDS_PAGE

10. http://elchem.kaistac.kr/chem-ed

# - file:///F:/electrophorsis/Capillary%20Electrophoresis%20-%20ChemWiki.htm

Li, Sam. Capillary Electrophoresis: Principles, Practice, and Applications. Journal of Chromatography Library;Elsevier Science Publishers: The Netherlands, 1992; Vol 52

gels are fun to run

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