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Transcript of Kariuki practical report
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.
REFERENCES
Arber W., Cinn S. (1969) DNA modification and restriction: Annual Review Biochemistry 38:
467-500
Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., Struhl, K.,
eds (2002) Short Protocols in Molecular Biology, 5th ed. John Wiley & Sons, New York.
Cohen, S. A. Chang (1972) Non-chromosomal antibiotic resistance in bacteria: Genetic
engineering of E.coli: R-factor DNA proceeding of National Academy of Sciences
Griffith, F. (1928) The significance of pneumococcal types. Journal of Hygiene V 27: 113-159
Kruger, D.H., Brickle T (1983) Bacteriophage survival; Multiple mechanisms for avoiding the
Deoxyribonucleic Acid Restriction Systems of their Host. Microbilogy Review 47: 345-360
Mandel M and A. Higa (1970) Calcium Dependent Bacteriophage DNA infection: Journal of
Molecular Biology 53 (1) 159-162
Sambrook, J. Russel (2001) Molecular cloning: A Laboratory Manual 3rd
ed, Cold Spring
Habour Laboratory Press: Cold Spring harbor, NY.
Steve Odongo (2011) IPMB General Laboratory Manual
Watson, James D. (2007). Recombinant DNA: genes and genomes: a short course. San
Francisco: W.H. Freeman