VRIJE UNIVERSITEIT BRUSSEL

INSTITUTE OF MOLECULAR BIOLOGY AND

BIOTECHNOLOGY

TITLE: DNA AND PROTEIN TECHNIQUES PRACTICALS REPORT

NAMES: KARIUKI SAMUEL MUNDIA

INSTRUCTOR: Steven Odongo

DATE OF SUBMISSION: 13/1/2012

A. DNA TECHNIQUES

1.0 CLONING NANOBODY® GENE

1.1 INTRODUCTION

The gene is the corner stone of most molecular biology techniques. It is possible today to

amplify a gene through insertion in another DNA molecule that serves as a vehicle or vector that

can effectively divide inside living cells (Watson, 2007). The most widely used vectors include

bacterial plasmids, Cosmids and phages. This is a recombinant DNA molecule that is then

inserted inside a prokaryotic or eukaryotic cell as host. As the host cell replicates, the vector

together with the inserted foreign DNA also replicates. Through this the foreign DNA becomes

amplified in number and further analysis can be performed.

Insights into recombinant DNA technology came from among others the observation of the

ability of the linear genome of lambda phage DNA to circularize when it enters the host bacteria

cell by Allan Campbell in 1962. Further analysis revealed that lamda phage had short regions of

single stranded DNA whose base sequences were complementary to each other at the end of its

linear genome referred to as „cos‟ sites (cohesive sites). Further insights came from

characterization of bacterial restriction/ modification systems by Salvador Luria and other phage

workers which became apparent bioengineering tools for creating cohesive ends through

restriction endonucleases.

Cloning vectors are the carrier of the DNA molecule and all vectors must have some important

features which include capability to independently replicate themselves and the foreign DNA

they carry, secondly, they must contain a number of restriction endonuclease cleavage sites

which are present only once in the vector. Thirdly they must carry a selectable marker usually

inform of an antibiotic resistance to distinguish host cells that carry the vector from host cells

that do not carry the vector. Finally, they must be relatively easy to recover from the host cell.

Transformation is the process of introducing the ligation mixture of recombinant and non-

recombinant DNA into the host bacterial cell. A mutant bioengineered strain of E.coli bacteria

deficient of restriction modification system is used. The traditional method of transformation

involve incubating the bacteria in a concentrated calcium salt overnight to make their membrane

leaky but a more efficient method involve heat shock treating or electroporation.

Amplification is done by Polymerase Chain Reaction, PCR developed by Kary Mullis in 1985.

With PCR, a single segment of DNA can be amplified a billion times in several hours. The

procedure is carried our entirely in vitro through three important processes. Since it is a DNA

polymerase reaction it requires a DNA template and a 3‟ OH provided by the DNA sample to be

amplified and the site-specific oligonucleotide primers respectively. The three steps of the

reaction are denaturation, annealing of the primers and extension of the primers. Denaturation is

the first step done by heating the double stranded DNA to make single stranded DNA template.

Annealing is done by cooling to allow the primers to bind to the appropriate complementary

strands. Primer extension is the last step and happens in the presence of magnesium ions and

DNA polymerase which extends the primers on both strands from 5‟ – 3‟ direction. Currently the

most popular DNA polymerase is Taq polymerase from thermophilic bacteria Thermus

aquaticus. Temperature variations is done using an instrument called a thermo cycler with the

capability of rapidly switching between the different temperatures that are required for PCR

reaction

Visualization of PCR products is done on a gel stained with a nucleic acid-specific fluorescent

compounds such as ethidium bromide or SYBR green.

2.0 MATERIALS AND METHODS

2.1 PLASMID ISOLATION

2.1.1 MATERIALS

Sterilized 2ml eppendorf tubes

Sterilized micropipette tips

Sterilized bacterial cultures

Ice in box

Centrifuge

Luria broth (LB) media (liquid)

Cell re-suspension solution (P1)

Cell lysis solution (P2)

Neutralization solution (P3)

Isopropanol (kept at -20oC)

70% (v/v) ethanol, (kept at -20oC)

Sterilized water

Waste beaker/ dissinfectants

E.coli call WK 1168 containing PHEN6c

2.1.2 PROCEDURE

An E. coli suspension incubated the previous night in 5ml LB containing ampicillin in a 50ml

tube at 370C was harvested and 1.5ml pelleted via centrifugation at 12,000xg for I minute. The

supernatant was discarded. The bacteria pellet was then re-suspended with 200µl of the re-

suspension solution (P1). Vortexing was done to thoroughly re-suspend the cells until

homogeneous.

The re-suspended cells were lysed by adding 200µl of the lysis solution (p2). The contents were

immediately mixed by gentle inversion 7 times until the mixture became clear and viscous. The

lysis reaction was not allowed to exceed 5 minutes. The cell debris was then precipitated by

adding 350µl of neutralizing solution (P3). The tube was gently inverted 6 times. The cell debris

was pelleted by centrifuging at 12,000xg for 10 minutes for cell debris, proteins, lipids, SDS and

chromosomal DNA to fall out of solution as cloudy, viscous precipitate.

The column was prepared by inserting the GenElute Miniprep Binding column into a provided

micro-centrifuge tube. 500µl of the column preparation solution was added to each miniprep

column and centrifuged at 12,000xg for 1 minute. The flow-through liquid was discarded. The

column preparation solution was meant to maximize the binding of DNA to the membrane

resulting in more consistent yields.

The cleared lysate from neutralization reaction above was transferred to the prepared column and

centrifuged at 12,000xg for one minute and the flow-through liquid discarded. 500µ of optional

wash solution was added to the column and centrifuged at 12,000xg for one minute and the flow-

through liquid discarded. 750µl of the diluted ethanol wash solution was added to the column

and centrifuged at 12,000xg for 1 minute. This column was step removes residual salt and other

contaminants introduced during the column load. The flow-through liquid is discarded and

centrifuged again at maximum speed for 2 minutes without additional wash to remove excess

ethanol.

To elute DNA, the column was transferred to a fresh column and 100µl of elution solution added

to the column followed centrifuging at 12,000xg for 1 minute.

The purified plasmid DNA was present in the elute and its concentration was determined using

the NanodropTM

. The elute was stored at -200C.

2.2 RESTRICTION ENDONUCLEASE DIGESTION OF NANOBODY GENE AND

PHEN6c

2.2.1 Materials

PCR fragment (Nanobody gene)

PHEN6c plasmid

10x O-buffer (fermentas)

PCR clean up kit-GenElute

Spectrophotometer/ NanodropTM

Water bath (370C)

Eco 911 (fermentas, 10 units/µl)

Pstl (fermentas, 10 units/µl)

Ethanol

Micro centrifuge

Micro centrifuge tubes

Molecular Biology water

2.2.2 Procedure

2.2.2.1 Digestion of vector for ligation and checking size.

Digestion was done by taking 10µq plasmid + 5µl O-buffer (fermentas) + 1µl PstI (fermentas, 10

units/µl) + 1µl Eco911 (fermentas, 10 units/µl) and topped up to 50µl. into another tube digest

for checking vector size was digested with only Pstl restriction enzyme and incubating for two

hours at 370C was performed. Checking digestion and vector size was done the following day

using 0.8% agarose. Digests for ligation were purified as follows.

The GenElute plasmid mini spin column is inserted into a provided collection tube. 0.5ml of the

column preparation solution was added to each mini spin column and centrifuged at 12,000xg for

1 minute. The elute was discarded. The column preparation solution maximizes binding of DNA

to the membrane resulting in more consistent yields. 250µl of the binding solution was added to

the 50µl of the plasmid DNA. The solution was transferred to the binding column and

centrifuged at 12,000xg for one minute. The elute was discarded but the collection tube was

retained.

The binding column was replaced into the collection tube.0.5ml of the diluted wash solution is

applied to the column and centrifuged at a maximum speed for one minute. The elute is

discarded but the collection tube retained. The column is replaced in the collection tube and

centrifuged at maximum speed for 2 minutes without any additional wash solution to remove

excess ethanol. The residual elute and as well as the collection tube were discarded. The column

was then transferred to a fresh 2ml collection tube. 50µl of elution solution was applied to the

centre of the column and incubated at room temperature for 1 minute. The column was then

centrifuged at maximum speed for 1 minute. The Plasmid DNA was available in the elute and its

purity concentration was determined by NanodropTM

.

The same procedure was followed by the other group to purify the PCR fragment (nanobody

gene).

2.3 LIGATION

2.3.1 Materials

Eppendorfs

Digested vector and PCR fragment

Water bath

dH2O

T4DNA ligase (5units/µl)

10 ligation buffer

2.3.2 Procedure

Ligation was done by mixing 50ng vector + 50ng PCR fragment (1:1) + 2µl 10x ligation buffer +

1µl T4 DNA ligase (5units/µl) and filled up with dH2O until the end volume was 20µl. All the

three tubes were incubated for 1 hour at room temperature.

2.4 HEAT SHOCK TRANSFORMATION

2.4.1 Generation of CaCl2 competent E. coli cells

2.4.1.1 Materials

Reagents

LB medium

Sterile ice cold 0.1M MgCl2

Sterile ice cold 0.1 100% glycerol

Fresh E.coli WK6 403 strain

Equipment

50ml blue caps

Sterile 1.5ml eppendorfs

Shaking flask with baffles

Cooled centrifuge for 50ml tubes

Laminar air flow

Spectrophotometer and cuvettes

2.4.1.2 Procedures

Five milliliters of LB (without antibiotics) is prepared in one sterile 50ml tube. The tube is then

inoculated with a single colony of E.coli WK 403 from a fresh plate. It was the tube was

incubated at 370C. The culture was harvested the following day.

The following morning, 20ml LB was inoculated with 0.2ml of overnight culture. The bacteria

cells were grown to early log phase i.e. OD600nm in cuvette = 0.3 reading was obtained in 180

minutes. The 20 ml culture was collected in a 50ml cap falcon and put on ice for 10 minutes. The

cells were then pelleted for 7 minutes at 3000 rpm in an eppendorf centrifuge at 40C. The

supernatant was removed and the pellet washed with 10ml sterile ice cold 0.1MgCl2. It was then

centrifuged at 3000rpm in 40C in eppendorf centrifuge.

The supernatant was removed and the pellet washed with 10ml sterile ice cold 0.1M CaCl2. It

was then incubated for thirty minutes on ice before centrifuging for 7 minutes at 3000 rpm in 40C

eppendorf centrifuge. The supernatant was removed and 2ml sterile ice cold 0.1M CaCl2 added

on the bacteria as well as 0.3ml sterile ice cold 100% glycerol. The mixture was incubated for 30

minutes on ice. The bacteria was then aliquoted in 100µl in 0.5 epperndorf.

2.4.2 Transformation by heat shock.

2.4.2.1 Materials

Sterilized eppendorf tubes

Sterilized micropipette tips

LB-Amp-Glu agar plates

Water bath

Ice

Laminar flow

Shaking incubator LB media (liquid) and 15g/l agar plate

Sterilized water

Centrifuge

Glass spreader/Bunsen flame/ march stick

Component cells

2.4.2.2 Procedure

Cells were used directly after competent cell preparation where three of the 0.5ml aliquots

(100µl) were obtained. In the first aliquot, 0.5µg (2.5µl) of purified intact plasmid DNA was

added (for calculation transformation efficiency) and to the second aliquot, 10µl of unpurified

ligation (to screen colonies, by PCR transformed with pHEN6c plasmid ligated with nanobody

DNA, and for subsequent calculation of the size of nanobody DNA) was added. The third aliquot

was left without adding anything to act as a negative control. The solution was mixed by

pipetting up and down. The tubes were then incubated in ice for 30 minutes. The tubes were then

placed in warm baths at 420C for exactly 90 seconds and put back in ice for 2 minutes.

Luria Broth (1.5ml) was added to each tube and placed in an incubator at 370C for 60 minutes.

The cells were then plated on LB agar containing ampicillin. Using a pipette 100µl of each

transformation preparation is dispensed on two LB-AMP agar plates. The same was done for

control (untransformed cells). With a sterile lazy spreader the liquid on agar was spread until all

was absorbed in the medium. Once the plates were dry, they were incubated at 370C upside down

overnight.

The following day, after 20 hours, the numbers of colonies were counted on each of the plates

plated with cells transformed with the intact plasmid DNA. The average count obtained was used

for calculation of transformation efficiency. The plates transformed with the ligate were spared

for colony PCR.

The transformation efficiency was calculated.

2.5 POLYMERASE CHAIN REACTION (PCR)

2.5.1 Materials

Laminar air flow

Thermo cycler

PCR tubes

Oligonucleotides

Primers, 10xPCR

dH2O

ice

2.5.2 Procedure

The following master mix was prepared on ice in a laminar flow chamber and since it was the

master mix for one tube, each measured was calculated for 6 tubes and put in the 7th

tube.

10x PCR buffer 5µl

dNTP (10mM total) 1µl

FP primer (20µM) 1µl

RP primer (20µM) 1µl

Taq DNA polymerase 0.25µl

dH2O 41.75µl

50µl

Three colonies are randomly picked using a sterile pipette tip from a test plate and one from each

of the control plates and dipped in separate tubes with 50µl master mix. The last tube served as a

negative control i.e. no template or colony added. The tips were left in the tubes for 15 minutes

and ticked away with care. The tubes were then closed and placed in the thermo cycler

programmed as below.

Program: pre-cycle 950C 3mins

28 cycle 940C 30sec

570C 30sec

720C 45sec

Post-cycle 720C 10mins

40C “until take out”

2.6 ANALYSIS OF DNA BY ELECTROPHORESIS BY AGAROSE GEL

ELECTROPHORESIS

2.6.1 Materials

gloves

electrophoresis cuves

small plastic transparent gel holder

2 black gel borders

1% (w/v) agarose gel in 1xTBE kept at 60oC in oven

TBE 1x buffer from 10x stock.

Ethidium bromide (EtBr)

20µl micropipettes + tips

DNA loading buffer 6x (to increase the density of samples so that they sink to the bottom

of slots/ wells)

Smart ladder kept at 4oC

Goggles

2.6.2 Procedure

The gloves were put on and the gel holder prepared by putting the gel holder to its edge borders.

It was tightened to avoid leakage of the gel. The combs were then put in place with the tips

pointing downwards on the gel holder.

The stock of 1% (w/v) agarose gel in 1xTBE (20g agarose in 2l 1x TBE) was taken and kept in

an oven at 60oC. The gel was then poured to make the cast on the gel holder.

Seven microliters of EtBr was mixed by stirring with the pipette tip while the gel was still liquid.

All air bubbles in the gel were removed by moving back and forward the comb. The gel was left

to solidify until a milky appearance was observed.

The border support was removed and the transparent gel holder with gel placed in the cuve. The

combs were removed and 1xTBE Buffer poured to completely cover the gel holder.

The prepared PCR samples and smart ladder were loaded very carefully into the slots. 16µl of

negative control was mixed with 10µl of loading buffer 6x and loaded into the second slot. 16µl

of PCR sample was mixed with 10µl of loading buffer 6x and loaded into the third, fourth and

fifth slots. The positive control was added into the sixth slot. 5µl of smart ladder was loaded into

the first slot.

The lid of the cuve was put and the apparatus connected to positive (red) and negative (black)

current. The voltage was put at 125v for one hour and when started air bubbles were observed

mounting at the top left corner under the lid. The current automatically went off and the power

supply was shut down and the lid taken off. The gel was loosen with care at the borders with a

sharp cutter. It was taken carefully from the gel holder and looked at by putting it in the UV light

booth.

The machine was put ON and bands of fluorescence were visible to the eye. The big cover with

camera was put on top instead of the plastic cover lid and the picture made. The image screen

and the printer were switched on. On the screen there were + and – button for sharpening the

images. A sharp print out was made.

Molecular weight of DNA after Gel electrophoresis was determined.

3.0 RESULTS

3.1 Plasmid isolation

Plasmid concentration obtained from nanoDrop™ was 151.5ng/µl = 0.151.5µg/µl

Yield = 0.1515*50µl = 7.575µg

3.2 Restriction endonuclease and transformation efficiency

Nanobody gene concentration = 33.10ng/µl

pHEN6c plasmid concentration = 119.8ng/µl

3.3 Transformation efficiency

colony number= 766cfu

Transformation efficiency was done as follows:

Amount of DNA= 0.5µg in 10µl added to 100µl cells.

Concentration of DNA in solution was therefore = 0.5/110 =0.004545µg/µl

Later, 1500µl LB added during phenotypic expression,

New concentration=0.5/1610= 3.106x10-4

µg/µl.

After, 100µl of culture added to each of the plates, therefore amount of intact plasmid DNA

plated on each plate was calculated as:

3.106x10-4

x100 = 3.106x10-2

µg

Amount of DNA plated (µg/ml) = 3.106x10-2

µg/100µl = 3.106x10-2

/0.1ml

= 3.106x10-1

µg/ml

Transformation efficiency =

=

= 2047

cfu/µg/ml

˜ 2.048x105 transformants per microgram

3.4 Gel electrophoresis and molecular weight calculation

3.4.1 Molecular weight of the Vector

Figure 1: Results of gel electrophoresis showing migration of the vector on agarose gel.

Table 1: Showing the relative distances of migration of the vector and log of molecular weight.

Migration of

standards(cm)

Relative distances

(X)

Molecular Weight of

standard.

Log molecular

weight (Y)

3.2 0.28 10000 4

3.5 0.3 8000 3.9

3.7 0.32 6000 3.8

4 0.35 5000 3.7

4.3 0.37 4000 3.6

4.7 0.41 3000 3.5

5 0.44 2500 3.4

5.3 0.46 2000 3.3

5.8 0.51 1500 3.2

6.7 0.59 1000 3

7.1 0.63 800 2.9

7.7 0.67 600 2.8

8.5 0.75 400 2.6

9.7 0.85 200 2.3

Sample

Migration=4.3cm

0.37

Dye migration distance=11.4cm

4000

3000

Figure 2: Graph of log of molecular weights versus relative distances for the calculation of molecular weight of the vector

Molecular weight of the vector

Equation of the line = y= -2.8582x+4.7005

X = 0.37

Therefore, y= -2.8582*0.37+4.7005 = 3.643

Antilog 2.643 =4395bp

3.4.2 Molecular weight of the Nanobody

Figure 3: Results of gel electrophoresis showing migration of the Nanobody on agarose gel.

y = -2.8582x + 4.7005 R² = 0.9911

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

0 0.2 0.4 0.6 0.8 1

log

of

std

mo

lecu

lar

we

igh

t

Relative distance (cm)

Vector molecular weight

400bp

600bp

Table 2: Showing the relative distances migration of Nanobody and log of molecular weight.

Migration of

standards(cm)

Relative distances

(X)

Molecular Weight of

standard.

Log molecular

weight (Y)

3.3 0.25 10000 4

3.7 0.28 8000 3.9

4 0.31 6000 3.8

4.4 0.34 5000 3.7

4.7 0.36 4000 3.6

5.2 0.4 3000 3.5

5.5 0.42 2500 3.4

6 0.46 2000 3.3

6.7 0.52 1500 3.2

8 0.62 1000 3

8.6 0.66 800 2.9

9.4 0.72 600 2.8

10.7 0.82 400 2.6

12 0.92 200 2.3

Sample

Migration=9.4cm

0.72

Dye migration distance=11.4cm

Figure 4: Graph of log of molecular weights versus relative distances for the calculation of molecular weight of the Nanobody.

y = -2.4091x + 4.504 R² = 0.9863

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

0 0.2 0.4 0.6 0.8 1

Log

of

mo

lecu

lar

we

igh

t o

f st

and

ard

Relative distances (cm)

Nanobody molecular weight

Molecular weight of Nanobody

Equation of the line = y=-2.4091x+4.504

X= 0.72

Therefore by substitution

Y= -2.4091*0.72+4.504 = 2.769

Antilog of 2.769 = 588bp

Results of colony PCR

4.0 DISCUSSION

Transformation is a technique used to introduce a plasmid inside a bacteria cell and to use the

bacteria to amplify this plasmid in-order to produce large quantities of it. It can be traced back to

1928 with Fredrick Griffith‟s experiment using Sreptococcus pneumonia, a bacteria that causes

respiratory tract infections (Griffith, 1928). Morton Mandel and Akiko Higa in 1970 showed that

Escherichia coli K12, a strain that is not naturally transformable, could be made competent to

take up DNA from bacteriophage lamda (Mandel and Higa, 1970). Thereafter Cohen and Chan

showed that circular, nicked circular or sheared DNA could be assimilated by bacteria cells and

recovered in covalently circular form (Cohen and Chang, 1972). Laboratories have since then

Lad

der

Liga

te

improved these earlier experiments to come up with more efficient transformation capability. In

our experiment we use an improved strain of WK403. To improve the efficiency, MgCL2 was

used to provide the divalent cation and the cells were used after they were in their log phase of

growth. This is because rapidly growing cells in their early log phase are very susceptible to

transformation. This was done by incubating the bacteria with LB media until an OD600nm of 0.4

was obtained. Further, the efficiency was enhanced by make our cells competent with CaCl2.

This serves to prevent unfavorable interactions between the incoming DNA and the polyanions

on the surface of the bacteria. Incubation on ice for one hour helped to allow increased

interaction of the calcium ions and the negative components of the cell. The change in

temperature in heat shock treatment at 42oC served to alter the fluidity of the semi-crystalline

membrane state achieved at 0oC thus allowing the DNA to enter through the zone of adhesion.

The competency of the stock competent cells is done by calculating the number of colonies of

bacteria produced per microgram of DNA added. An excellent preparation has a competence of

108 cells per microgram while a poor one has 10

4cells/µg. Amazingly, our transformation

efficiency was within the range, i.e. 105 cells/µg.

Restriction enzymes are used to cut double stranded DNA at regions called the restriction site. It

is believed that bacteria have evolved to include restriction enzymes to survive virus attack

(Arber and Cinn, 1969). The hosts DNA is protected against the endonuclease activity by

methylation of its bases (Kruger and Brickle, 1983). In our experiment, both the vector and the

nanobody PCR product were restricted using two enzymes, Eco911 and Pstl. Ligation of the two

was done followed by transformation.

Gel electrophoresis is a technique used to separate a population of nucleic acids in molecular

biology lab based on size and electric charge. DNA moves through the pores of the gel through

sieving phenomenon. Shorter molecules move faster and longer than larger one (Sambrook and

Russel, 2001). The results of gel electrophoresis were used to calculate the molecular weight of

both the vector, the nonobody PCR product and the ligated (recombinant plasmid). The

molecular weight of the vector was 4395bp, and that of Nanobody had 588bp.

B. PROTEIN TECHNIQUES

1.0 INTRODUCTION

Proteins are gene products, the results of DNA transcription and eventual translation (central

Dogma) and folding to fun functional units. Several methods are employed in study of proteins.

These include genetic methods, isolating and purifying proteins, and methods of characterizing

the structure and functions of these proteins.

Protein purification is a crucial step while working with proteins and scientists should be able to

isolate and purify proteins of interest so their conformations, substrate specificities, reaction with

other ligands and specific activities can be studied. Several protein purification methods are used

which include chromatographic methods, ion exchange, size exclusion or gel filtration, affinity

chromatography.

Protein detection can be done by among others, immune blotting, BCA assay, western blotting,

spectrophotometry and enzyme assay.

2.0 ENZYME LINK IMMUNOSORBENT ASSAY (ELISA)

2.1 Introduction

ELISA is a plate based assay designed for detecting and quantifying substances such as peptides,

proteins, antibodies and hormones. An antigen must be immobilized to a solid surface and then

complexed with an antibody that is linked to an enzyme. Detection is accomplished by assessing

the conjugated enzyme activity via incubation with a substrate to produce a measurable product.

The most crucial element of the detection is a highly specific antibody- antigen interaction.

ELISA is generally performed in 96-well or 384-well polystyrene plates that passively bind the

antigen or protein.

ELISA can be done with a number of modifications to the basic procedure. The key step,

immobilization of antigen of interest can be accomplished by direct adsorption to the assay plate

or indirectly via a capture antibody that has been attached to the plate. The antigen is then

detected either directly via primary antibody or indirectly via secondary antibody. The most

powerful ELISA assay is “sandwich.” The analyte to be measured is bound between two primary

antibodies, the capture and detection antibody. It is sensitive and robust.

2.2 Objective:

To investigate presence of anti-Variant Surface Glycoprotein (VSG) antibodies in a serum

sample obtained from alpaca immunized with VSG antigen.

2.3 materials

Soluble VSG protein (coat plate at 1µg/ ml)

Nunc 96 well flat bottom plate maxisorp

Serum fraction diluted 1/10 in PBS (vortex)

PBS

AP- blot buffer+ alkaline phosphatase substrate at 2mg/ml

Blocking milk: 1%milk powder in PBS

Multichannel pipette 100µl + yellow tips

0.1% (v/v) Tween 20 in phosphate buffered saline (PBS-T)

Rabbit α-Ilama IgG 1/100

1/2000 α-Rabbit AP

Spectrophotometer

2.4 Procedure

The plate (6 wells) was coated with 100µl of 1µl of VSG antigen overnight at 4oC. The

following day, the overnight coating was thrown away and the wells rinsed 3 times with 300µl

0.1% PBS-T. The wells were then blocked with 200µl blocking milk 1% in PBS for one hour.

The wells were then washed five times using 0.1%PBS-T

The three serum samples were then diluted 1/10. In all rows except the last, 100µl of PBS was

added followed by 1/10 dilute serum in the order, positive control, test sample, negative control

and the last two left blank. The plate was incubated for one hour at room temperature before

washing five times with 0.1% PBS-T.

In each well 100µl of Rabbit α-Ilama IgG 1/1000 was added followed by incubation for 1 hour at

room temperature. 100µl/ well of α-Rabbit-HRP diluted to 1/1000 was added followed by

incubation for one hour after which the wells were washed 4x with 0.1%PBS-T. In each well

100µl of 1-step TM Slow TMB-ELISA substrate was added and the reaction stopped with 100µl

1mH2SO4 after color development was observed. The plate was then read on a

spectrophotometer at 450nm and the absorbance recorded.

2.5 Results

The spectrophotometer output produced the following absorbance table.

Table 3: Average of absorbances

Ist replica 2nd

replica average

Positive control 1.682 1.934 1.808

Test sample 1.263 1.571 1.417

Negative control 0.054 0.058 0.056

Blank 0.056 0.061 0.0585

Results of absorbance minus the background interference

Table 4: Average absorbance less background interference

Ist replica 2nd

replica average

Positive control 1.626 1.873 1.7495

Test sample 1.207 1.51 1.3585

Negative control -0.002 -0.003 -0.0025

2.6 Discussion

The original trypanosome sample provided had a concentration of 3.03mg/ml. To obtain an

aliquot of 700µl (6 wellsx100µl+100µl excess), the concentration was first brought down from

3.03mg/ml through 100µl/ml then to required final concentration of 1µl/ml(working solution)

using the formula CiVi=CfVf. As well as getting the right working solution concentration, this

procedure also help to magnify the volume from the small volume provided.

In ELISA an unknown amount of antigen is fixed and a specific antibody is applied in order to

bind to the substrate. This antibody is linked to an enzyme in which the last step involves

reaction with a substrate to produce a detectable signal. The sample with an unknown amount of

antigen is immobilized on a solid support either non-specifically by adsorption directly on the

plate or specifically via capture by another specific antibody that binds the solid support as in

sandwich ELISA. The immobilized antigen is the detected by another antibody linked to an

enzyme or itself may be detected by another secondary antibody linked to an enzyme as in bio

conjugation ELISA.

Washing after every step is mandatory as well as blocking to remove background coloration of

the polystyrene micro-titer plate wells

3.0 DETERMINATION OF PROTEIN CONCENTRATION

3.1 Introduction

Biochemical analysis of proteins relies on accurate quantification of protein concentration.

Several methods are used for quantification of protein concentration, Bradford and BCA are

however the routinely used methods.

BCA assay is a two-step assay, in which Cu2+ is first reduced to Cu1+ forming a complex with

protein amide bonds (Biuret reaction). Secondly, bicinchoninic acid (BCA) forms a purple

complex with Cu1+ which is detectable at 562nm. The assay is sensitive but slow unless heated.

3.2 Objective:

To determine the concentration of purified protein(Nanobody) by Bicinchoninic Acid (BCA)

Assay.

3.3 Requirements

3.3.1 Materials

Eppendorf tubes

Micropipette tips

Distilled water

Bovine Serum Albumin (BSA)

Incubator at 37oC

Pierce Protein Assay Kit

96-wells flat-bottom plates

Spectrophotometer

3.3.2 Recipes

Reagent A, 1L

10g BCA(1%)

20g Sodium carbonate (Na2CO3.2H2O)-2%

1.6g Sodium tartrate (Na2C4H4O62H2O)-0.16%

4g Sodium Hydroxide (NaOH)-0.4%

9.5g NaHCO3 (0.95%)

Distilled water to 1L

pH adjusted to 11.25

Reagent B 50ml

2gCuSO4.5H2O (4%)

Distilled water to 50ml

3.4 Procedure

3.4.1 Preparation of working reagent

Twenty five parts of reagent A, 24 parts of reagent B and one part of reagent C were mixed

together. The amount of working reagent required for each sample was 1ml for the test tube

procedure and 150µl for the micro-assay plate procedure. Since the test tube procedure was

already performed in lieu of the practical, micro-assay procedure was performed. Consequently

44 wells of the micro-titer plate were filled with 150µl each working reagent.

3.4.2 Preparation of BSA standard

The available concentration of the BSA standard was 2mg/ml in a 1ml volume. This stock

solution was diluted to 80µg/ml to increase the volume to a draw able quantity. A duplicate of

tapering concentrations (4-40µg/ml) were prepared with the same diluent (PBS) used to dilute

the sample.

3.4.3 Preparation of sample

A two fold serial dilutions of the sample were prepared i.e. 300µl of PBS and 300µl sample and

added into the 20 wells and two blanks left.

Table 5: BSA concentrations and sample serial dilutions

1 2 3 4 5 6 7 8 9 10 11 12

Standard

µg/ml

A 40 36 32 28 24 20 16 12 8 4 Blank

B 40 36 32 28 24 20 16 12 8 4 Blank

sample C Conc 1/2 1/4 1/8 1/16 1/32 1/64 1/128 1/256 1/512 Blank

C Conc 1/2 1/4 1/8 1/16 1/32 1/64 1/128 1/256 1/512 Blank

E

3.4.4 Micro-plate procedure

The linear working ranges of 4-40µl/ml were used. 150µl of each working standard were

pipetted into a micro-well plate. 150µl of the working reagent were added to each well and

mixed thoroughly on a plate shaker for 30 seconds. The plate was the covered and incubated at

37oC for two hours before cooling to room temperature. The absorbance was then measured at

562 of all the samples on the plate reader and the absorbance value of the blank subtracted from

the readings of the standards and the unknowns. The blank-corrected 562nm reading for each

standard vs. its concentration was plotted. The protein concentration of each unknown was

determined from the calibration plot.

3.5 Results

Absorbances

Table 6: Absorbance of both the sample and the BSA standard.

1 2 3 4 5 6 7 8 9 10 11

std 0.406 0.390 0.347 0.316 0.286 0.255 0.231 0.186 0.155 0.142 0.099

0.416 0.399 0.357 0.326 0.295 0.254 0.223 0.195 0.164 0.145 0.108

sample 1.872 1.186 0.721 0.432 0.264 0.177 0.132 0.119 0.107 0.129 0.107

1.857 1.195 0.737 0.450 0.271 0.184 0.135 0.126 0.110 0.111 0.114

Table 7: Blank-corrected absorbance reading.

1 2 3 4 5 6 7 8 9 10

std 0.307 0.291 0.248 0.217 0.187 0.156 0.132 0.087 0.056 0.043

0.308 0.291 0.249 0.218 0.187 0.146 0.115 0.087 0.056 0.037

sample 1.765 1.079 0.614 0.325 0.157 0.07 0.025 0.012 0 0.022

1.743 1.081 0.623 0.336 0.157 0.07 0.021 0.012 -0.004 -0.003

Table 8: BSA standard and their mean absorbances

1 2 3 4 5 6 7 8 9 10

BSA

std

µg/ml

40 36 32 28 24 20 16 12 8 4

Mean

Ab595

0.3075 0.291 0.2485 0.2175 0.187 0.151 0.1235 0.087 0.056 0.04

Table 9: Sample dilution and mean absorbance

1 2 3 4 5 6 7 8 9 10

Sample

dilution

Conc 1/2 1/4 1/8 1/16 1/32 1/64 1/128 1/256 1/512

Mean

Ab595

1.754 1.08 0.6185 0.3305 0.157 0.07 0.023 0.012 -0.002 -

0.0015

Figure 5: Graph showing relationship between Absorbance and BSA standard concentrations for the calculation of sample concentration.

Protein concentration calculated from the graph e.g. for 1/16 dilution

Y= 0.0078x-0.0017

0.157=0.0078x-0.0017

0.1587=0.0078x

X= 20.35µg/ml

Concentration at 1/16 is 20.35µl/ml and therefore the concentration of the undiluted protein

equals 16x 20.35 = 325.6µg/ml

y = 0.0078x - 0.0017 R² = 0.9962

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0 5 10 15 20 25 30 35 40 45

Ab

sorb

ance

(n

m)

BSA concentration (µg/ml)

Absorbance of standard BSA

3.6 Discussion.

The absorbance at 1/16 was used since it was the first to fall within the range of the standard.

BCA method of determination of protein concentration relies on plotting of a standard curve.

The absorbance of known protein concentrations are used to plot this curve. A comparison of the

absorbance of the known and unknown protein concentrations is then done. Ordinarily, Bovine

Serum Albumin (BSA) is used to make the standard curve. This is probably due to its wide

availability in powder form and can therefore be weighed conveniently in the lab. In addition it

dissolves in water to form a colorless solution which reacts with coomassie blue to form a deep

blue addition product.

Serial dilution of the sample of protein is done because you do not know the concentration of it

and it could be less of more than your standard and therefore serial dilution is done to increase

the likelihood that you will be able to produce a sample with absorbance that falls within the

range of the standard curve.

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Kruger, D.H., Brickle T (1983) Bacteriophage survival; Multiple mechanisms for avoiding the

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Sambrook, J. Russel (2001) Molecular cloning: A Laboratory Manual 3rd

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