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  • 2. A. DNA TECHNIQUES1.0 CLONING NANOBODY® GENE1.1 INTRODUCTIONThe gene is the corner stone of most molecular biology techniques. It is possible today toamplify a gene through insertion in another DNA molecule that serves as a vehicle or vector thatcan effectively divide inside living cells (Watson, 2007). The most widely used vectors includebacterial plasmids, Cosmids and phages. This is a recombinant DNA molecule that is theninserted inside a prokaryotic or eukaryotic cell as host. As the host cell replicates, the vectortogether with the inserted foreign DNA also replicates. Through this the foreign DNA becomesamplified in number and further analysis can be performed.Insights into recombinant DNA technology came from among others the observation of theability of the linear genome of lambda phage DNA to circularize when it enters the host bacteriacell by Allan Campbell in 1962. Further analysis revealed that lamda phage had short regions ofsingle stranded DNA whose base sequences were complementary to each other at the end of itslinear genome referred to as „cos‟ sites (cohesive sites). Further insights came fromcharacterization of bacterial restriction/ modification systems by Salvador Luria and other phageworkers which became apparent bioengineering tools for creating cohesive ends throughrestriction endonucleases.Cloning vectors are the carrier of the DNA molecule and all vectors must have some importantfeatures which include capability to independently replicate themselves and the foreign DNAthey carry, secondly, they must contain a number of restriction endonuclease cleavage siteswhich are present only once in the vector. Thirdly they must carry a selectable marker usuallyinform of an antibiotic resistance to distinguish host cells that carry the vector from host cellsthat 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 bacteriadeficient of restriction modification system is used. The traditional method of transformationinvolve incubating the bacteria in a concentrated calcium salt overnight to make their membraneleaky 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. Theprocedure is carried our entirely in vitro through three important processes. Since it is a DNApolymerase reaction it requires a DNA template and a 3‟ OH provided by the DNA sample to beamplified and the site-specific oligonucleotide primers respectively. The three steps of thereaction are denaturation, annealing of the primers and extension of the primers. Denaturation isthe 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
  • 3. strands. Primer extension is the last step and happens in the presence of magnesium ions andDNA polymerase which extends the primers on both strands from 5‟ – 3‟ direction. Currently themost popular DNA polymerase is Taq polymerase from thermophilic bacteria Thermusaquaticus. Temperature variations is done using an instrument called a thermo cycler with thecapability of rapidly switching between the different temperatures that are required for PCRreactionVisualization of PCR products is done on a gel stained with a nucleic acid-specific fluorescentcompounds such as ethidium bromide or SYBR green.2.0 MATERIALS AND METHODS2.1 PLASMID ISOLATION2.1.1 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 PHEN6c2.1.2 PROCEDUREAn E. coli suspension incubated the previous night in 5ml LB containing ampicillin in a 50mltube at 370C was harvested and 1.5ml pelleted via centrifugation at 12,000xg for I minute. Thesupernatant 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 untilhomogeneous. The re-suspended cells were lysed by adding 200µl of the lysis solution (p2). The contents wereimmediately mixed by gentle inversion 7 times until the mixture became clear and viscous. Thelysis reaction was not allowed to exceed 5 minutes. The cell debris was then precipitated byadding 350µl of neutralizing solution (P3). The tube was gently inverted 6 times. The cell debris
  • 4. was pelleted by centrifuging at 12,000xg for 10 minutes for cell debris, proteins, lipids, SDS andchromosomal DNA to fall out of solution as cloudy, viscous precipitate.The column was prepared by inserting the GenElute Miniprep Binding column into a providedmicro-centrifuge tube. 500µl of the column preparation solution was added to each miniprepcolumn and centrifuged at 12,000xg for 1 minute. The flow-through liquid was discarded. Thecolumn preparation solution was meant to maximize the binding of DNA to the membraneresulting in more consistent yields.The cleared lysate from neutralization reaction above was transferred to the prepared column andcentrifuged at 12,000xg for one minute and the flow-through liquid discarded. 500µ of optionalwash 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 columnand centrifuged at 12,000xg for 1 minute. This column was step removes residual salt and othercontaminants introduced during the column load. The flow-through liquid is discarded andcentrifuged again at maximum speed for 2 minutes without additional wash to remove excessethanol.To elute DNA, the column was transferred to a fresh column and 100µl of elution solution addedto the column followed centrifuging at 12,000xg for 1 minute.The purified plasmid DNA was present in the elute and its concentration was determined usingthe NanodropTM. The elute was stored at -200C.2.2 RESTRICTION ENDONUCLEASE DIGESTION OF NANOBODY GENE ANDPHEN6c2.2.1 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
  • 5. 2.2.2 Procedure2.2.2.1 Digestion of vector for ligation and checking size.Digestion was done by taking 10µq plasmid + 5µl O-buffer (fermentas) + 1µl PstI (fermentas, 10units/µl) + 1µl Eco911 (fermentas, 10 units/µl) and topped up to 50µl. into another tube digestfor checking vector size was digested with only Pstl restriction enzyme and incubating for twohours at 370C was performed. Checking digestion and vector size was done the following dayusing 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 thecolumn preparation solution was added to each mini spin column and centrifuged at 12,000xg for1 minute. The elute was discarded. The column preparation solution maximizes binding of DNAto the membrane resulting in more consistent yields. 250µl of the binding solution was added tothe 50µl of the plasmid DNA. The solution was transferred to the binding column andcentrifuged at 12,000xg for one minute. The elute was discarded but the collection tube wasretained.The binding column was replaced into the collection tube.0.5ml of the diluted wash solution isapplied to the column and centrifuged at a maximum speed for one minute. The elute isdiscarded but the collection tube retained. The column is replaced in the collection tube andcentrifuged at maximum speed for 2 minutes without any additional wash solution to removeexcess ethanol. The residual elute and as well as the collection tube were discarded. The columnwas then transferred to a fresh 2ml collection tube. 50µl of elution solution was applied to thecentre of the column and incubated at room temperature for 1 minute. The column was thencentrifuged at maximum speed for 1 minute. The Plasmid DNA was available in the elute and itspurity concentration was determined by NanodropTM.The same procedure was followed by the other group to purify the PCR fragment (nanobodygene).2.3 LIGATION2.3.1 Materials  Eppendorfs  Digested vector and PCR fragment  Water bath  dH2O  T4DNA ligase (5units/µl)  10 ligation buffer
  • 6. 2.3.2 ProcedureLigation 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 thethree tubes were incubated for 1 hour at room temperature.2.4 HEAT SHOCK TRANSFORMATION2.4.1 Generation of CaCl2 competent E. coli cells2.4.1.1 MaterialsReagents  LB medium  Sterile ice cold 0.1M MgCl2  Sterile ice cold 0.1 100% glycerol  Fresh E.coli WK6 403 strainEquipment  50ml blue caps  Sterile 1.5ml eppendorfs  Shaking flask with baffles  Cooled centrifuge for 50ml tubes  Laminar air flow  Spectrophotometer and cuvettes2.4.1.2 ProceduresFive milliliters of LB (without antibiotics) is prepared in one sterile 50ml tube. The tube is theninoculated with a single colony of E.coli WK 403 from a fresh plate. It was the tube wasincubated at 370C. The culture was harvested the following day.The following morning, 20ml LB was inoculated with 0.2ml of overnight culture. The bacteriacells were grown to early log phase i.e. OD600nm in cuvette = 0.3 reading was obtained in 180minutes. The 20 ml culture was collected in a 50ml cap falcon and put on ice for 10 minutes. Thecells were then pelleted for 7 minutes at 3000 rpm in an eppendorf centrifuge at 40C. Thesupernatant was removed and the pellet washed with 10ml sterile ice cold 0.1MgCl2. It was thencentrifuged at 3000rpm in 40C in eppendorf centrifuge. The supernatant was removed and the pellet washed with 10ml sterile ice cold 0.1M CaCl 2. Itwas then incubated for thirty minutes on ice before centrifuging for 7 minutes at 3000 rpm in 40Ceppendorf centrifuge. The supernatant was removed and 2ml sterile ice cold 0.1M CaCl2 added
  • 7. on the bacteria as well as 0.3ml sterile ice cold 100% glycerol. The mixture was incubated for 30minutes on ice. The bacteria was then aliquoted in 100µl in 0.5 epperndorf.2.4.2 Transformation by heat shock. 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 cells2.4.2.2 ProcedureCells 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 wasadded (for calculation transformation efficiency) and to the second aliquot, 10µl of unpurifiedligation (to screen colonies, by PCR transformed with pHEN6c plasmid ligated with nanobodyDNA, and for subsequent calculation of the size of nanobody DNA) was added. The third aliquotwas left without adding anything to act as a negative control. The solution was mixed bypipetting up and down. The tubes were then incubated in ice for 30 minutes. The tubes were thenplaced 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 eachtransformation preparation is dispensed on two LB-AMP agar plates. The same was done forcontrol (untransformed cells). With a sterile lazy spreader the liquid on agar was spread until allwas absorbed in the medium. Once the plates were dry, they were incubated at 370C upside downovernight.The following day, after 20 hours, the numbers of colonies were counted on each of the platesplated with cells transformed with the intact plasmid DNA. The average count obtained was usedfor calculation of transformation efficiency. The plates transformed with the ligate were sparedfor colony PCR.The transformation efficiency was calculated.
  • 8. 2.5 POLYMERASE CHAIN REACTION (PCR)2.5.1 Materials  Laminar air flow  Thermo cycler  PCR tubes  Oligonucleotides  Primers, 10xPCR  dH2O  ice2.5.2 ProcedureThe following master mix was prepared on ice in a laminar flow chamber and since it was themaster mix for one tube, each measured was calculated for 6 tubes and put in the 7th tube.10x PCR buffer 5µldNTP (10mM total) 1µlFP primer (20µM) 1µlRP primer (20µM) 1µlTaq DNA polymerase 0.25µldH2O 41.75µl 50µlThree colonies are randomly picked using a sterile pipette tip from a test plate and one from eachof the control plates and dipped in separate tubes with 50µl master mix. The last tube served as anegative control i.e. no template or colony added. The tips were left in the tubes for 15 minutesand ticked away with care. The tubes were then closed and placed in the thermo cyclerprogrammed as below.Program: pre-cycle 950C 3mins 28 cycle 940C 30sec 570C 30sec 720C 45sec Post-cycle 720C 10mins 40C “until take out”
  • 9. 2.6 ANALYSIS OF DNA BY ELECTROPHORESIS BY AGAROSE GELELECTROPHORESIS2.6.1 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  Goggles2.6.2 ProcedureThe 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 tipspointing 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 inan 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 leftto solidify until a milky appearance was observed.The border support was removed and the transparent gel holder with gel placed in the cuve. Thecombs 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 ofnegative control was mixed with 10µl of loading buffer 6x and loaded into the second slot. 16µlof PCR sample was mixed with 10µl of loading buffer 6x and loaded into the third, fourth andfifth slots. The positive control was added into the sixth slot. 5µl of smart ladder was loaded intothe 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 observedmounting at the top left corner under the lid. The current automatically went off and the power
  • 10. supply was shut down and the lid taken off. The gel was loosen with care at the borders with asharp cutter. It was taken carefully from the gel holder and looked at by putting it in the UV lightbooth.The machine was put ON and bands of fluorescence were visible to the eye. The big cover withcamera was put on top instead of the plastic cover lid and the picture made. The image screenand the printer were switched on. On the screen there were + and – button for sharpening theimages. A sharp print out was made.Molecular weight of DNA after Gel electrophoresis was determined.3.0 RESULTS3.1 Plasmid isolationPlasmid concentration obtained from nanoDrop™ was 151.5ng/µl = 0.151.5µg/µlYield = 0.1515*50µl = 7.575µg3.2 Restriction endonuclease and transformation efficiencyNanobody gene concentration = 33.10ng/µlpHEN6c plasmid concentration = 119.8ng/µl3.3 Transformation efficiencycolony number= 766cfuTransformation 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/µlLater, 1500µl LB added during phenotypic expression,New concentration=0.5/1610= 3.106x10-4µg/µl.After, 100µl of culture added to each of the plates, therefore amount of intact plasmid DNAplated on each plate was calculated as:3.106x10-4 x100 = 3.106x10-2µgAmount of DNA plated (µg/ml) = 3.106x10-2µg/100µl = 3.106x10-2/0.1ml= 3.106x10-1µg/ml
  • 11. Transformation efficiency = = = 2047cfu/µg/ml˜ 2.048x105 transformants per microgram3.4 Gel electrophoresis and molecular weight calculation3.4.1 Molecular weight of the Vector 4000 3000Figure 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 Relative distances Molecular Weight of Log molecularstandards(cm) (X) standard. weight (Y)3.2 0.28 10000 43.5 0.3 8000 3.93.7 0.32 6000 3.84 0.35 5000 3.74.3 0.37 4000 3.64.7 0.41 3000 3.55 0.44 2500 3.45.3 0.46 2000 3.35.8 0.51 1500 3.26.7 0.59 1000 37.1 0.63 800 2.97.7 0.67 600 2.88.5 0.75 400 2.69.7 0.85 200 2.3Sample 0.37Migration=4.3cmDye migration distance=11.4cm
  • 12. Vector molecular weight 4.5 log of std molecular weight 4 y = -2.8582x + 4.7005 R² = 0.9911 3.5 3 2.5 2 1.5 1 0.5 0 0 0.2 0.4 0.6 0.8 1 Relative distance (cm)Figure 2: Graph of log of molecular weights versus relative distances for the calculation of molecular weight of the vectorMolecular weight of the vectorEquation of the line = y= -2.8582x+4.7005X = 0.37Therefore, y= -2.8582*0.37+4.7005 = 3.643Antilog 2.643 =4395bp3.4.2 Molecular weight of the Nanobody 600bp 400bpFigure 3: Results of gel electrophoresis showing migration of the Nanobody on agarose gel.
  • 13. Table 2: Showing the relative distances migration of Nanobody and log of molecular weight.Migration of Relative distances Molecular Weight of Log molecularstandards(cm) (X) standard. weight (Y)3.3 0.25 10000 43.7 0.28 8000 3.94 0.31 6000 3.84.4 0.34 5000 3.74.7 0.36 4000 3.65.2 0.4 3000 3.55.5 0.42 2500 3.46 0.46 2000 3.36.7 0.52 1500 3.28 0.62 1000 38.6 0.66 800 2.99.4 0.72 600 2.810.7 0.82 400 2.612 0.92 200 2.3Sample 0.72Migration=9.4cmDye migration distance=11.4cm Nanobody molecular weight 4.5 y = -2.4091x + 4.504 Log of molecular weight of standard 4 R² = 0.9863 3.5 3 2.5 2 1.5 1 0.5 0 0 0.2 0.4 0.6 0.8 1 Relative distances (cm)Figure 4: Graph of log of molecular weights versus relative distances for the calculation of molecular weight of theNanobody.
  • 14. Molecular weight of NanobodyEquation of the line = y=-2.4091x+4.504X= 0.72Therefore by substitutionY= -2.4091*0.72+4.504 = 2.769Antilog of 2.769 = 588bpResults of colony PCR Ladder Ligate4.0 DISCUSSIONTransformation is a technique used to introduce a plasmid inside a bacteria cell and to use thebacteria to amplify this plasmid in-order to produce large quantities of it. It can be traced back to1928 with Fredrick Griffith‟s experiment using Sreptococcus pneumonia, a bacteria that causesrespiratory tract infections (Griffith, 1928). Morton Mandel and Akiko Higa in 1970 showed thatEscherichia coli K12, a strain that is not naturally transformable, could be made competent totake up DNA from bacteriophage lamda (Mandel and Higa, 1970). Thereafter Cohen and Chanshowed that circular, nicked circular or sheared DNA could be assimilated by bacteria cells andrecovered in covalently circular form (Cohen and Chang, 1972). Laboratories have since then
  • 15. improved these earlier experiments to come up with more efficient transformation capability. Inour experiment we use an improved strain of WK403. To improve the efficiency, MgCL2 wasused to provide the divalent cation and the cells were used after they were in their log phase ofgrowth. This is because rapidly growing cells in their early log phase are very susceptible totransformation. This was done by incubating the bacteria with LB media until an OD600nm of 0.4was obtained. Further, the efficiency was enhanced by make our cells competent with CaCl 2.This serves to prevent unfavorable interactions between the incoming DNA and the polyanionson the surface of the bacteria. Incubation on ice for one hour helped to allow increasedinteraction of the calcium ions and the negative components of the cell. The change intemperature in heat shock treatment at 42oC served to alter the fluidity of the semi-crystallinemembrane 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 ofbacteria produced per microgram of DNA added. An excellent preparation has a competence of108 cells per microgram while a poor one has 104cells/µg. Amazingly, our transformationefficiency was within the range, i.e. 105 cells/µg.Restriction enzymes are used to cut double stranded DNA at regions called the restriction site. Itis 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 bymethylation of its bases (Kruger and Brickle, 1983). In our experiment, both the vector and thenanobody PCR product were restricted using two enzymes, Eco911 and Pstl. Ligation of the twowas done followed by transformation.Gel electrophoresis is a technique used to separate a population of nucleic acids in molecularbiology lab based on size and electric charge. DNA moves through the pores of the gel throughsieving phenomenon. Shorter molecules move faster and longer than larger one (Sambrook andRussel, 2001). The results of gel electrophoresis were used to calculate the molecular weight ofboth the vector, the nonobody PCR product and the ligated (recombinant plasmid). Themolecular weight of the vector was 4395bp, and that of Nanobody had 588bp.
  • 16. B. PROTEIN TECHNIQUES1.0 INTRODUCTIONProteins are gene products, the results of DNA transcription and eventual translation (centralDogma) 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 characterizingthe structure and functions of these proteins.Protein purification is a crucial step while working with proteins and scientists should be able toisolate and purify proteins of interest so their conformations, substrate specificities, reaction withother ligands and specific activities can be studied. Several protein purification methods are usedwhich include chromatographic methods, ion exchange, size exclusion or gel filtration, affinitychromatography.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 IntroductionELISA 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 thencomplexed with an antibody that is linked to an enzyme. Detection is accomplished by assessingthe 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 theantigen 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 plateor indirectly via a capture antibody that has been attached to the plate. The antigen is thendetected either directly via primary antibody or indirectly via secondary antibody. The mostpowerful ELISA assay is “sandwich.” The analyte to be measured is bound between two primaryantibodies, 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 serumsample obtained from alpaca immunized with VSG antigen.
  • 17. 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  Spectrophotometer2.4 ProcedureThe plate (6 wells) was coated with 100µl of 1µl of VSG antigen overnight at 4oC. Thefollowing day, the overnight coating was thrown away and the wells rinsed 3 times with 300µl0.1% 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-TThe three serum samples were then diluted 1/10. In all rows except the last, 100µl of PBS wasadded followed by 1/10 dilute serum in the order, positive control, test sample, negative controland the last two left blank. The plate was incubated for one hour at room temperature beforewashing 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 atroom temperature. 100µl/ well of α-Rabbit-HRP diluted to 1/1000 was added followed byincubation for one hour after which the wells were washed 4x with 0.1%PBS-T. In each well100µl of 1-step TM Slow TMB-ELISA substrate was added and the reaction stopped with 100µl1mH2SO4 after color development was observed. The plate was then read on aspectrophotometer at 450nm and the absorbance recorded.2.5 ResultsThe spectrophotometer output produced the following absorbance table.
  • 18. Table 3: Average of absorbances Ist replica 2nd replica averagePositive control 1.682 1.934 1.808Test sample 1.263 1.571 1.417Negative control 0.054 0.058 0.056Blank 0.056 0.061 0.0585Results of absorbance minus the background interferenceTable 4: Average absorbance less background interference Ist replica 2nd replica averagePositive control 1.626 1.873 1.7495Test sample 1.207 1.51 1.3585Negative control -0.002 -0.003 -0.00252.6 DiscussionThe original trypanosome sample provided had a concentration of 3.03mg/ml. To obtain analiquot of 700µl (6 wellsx100µl+100µl excess), the concentration was first brought down from3.03mg/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, thisprocedure 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 tobind to the substrate. This antibody is linked to an enzyme in which the last step involvesreaction with a substrate to produce a detectable signal. The sample with an unknown amount ofantigen is immobilized on a solid support either non-specifically by adsorption directly on theplate or specifically via capture by another specific antibody that binds the solid support as insandwich ELISA. The immobilized antigen is the detected by another antibody linked to anenzyme or itself may be detected by another secondary antibody linked to an enzyme as in bioconjugation ELISA.Washing after every step is mandatory as well as blocking to remove background coloration ofthe polystyrene micro-titer plate wells
  • 19. 3.0 DETERMINATION OF PROTEIN CONCENTRATION3.1 IntroductionBiochemical analysis of proteins relies on accurate quantification of protein concentration.Several methods are used for quantification of protein concentration, Bradford and BCA arehowever the routinely used methods.BCA assay is a two-step assay, in which Cu2+ is first reduced to Cu1+ forming a complex withprotein amide bonds (Biuret reaction). Secondly, bicinchoninic acid (BCA) forms a purplecomplex 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 Requirements3.3.1 Materials  Eppendorf tubes  Micropipette tips  Distilled water  Bovine Serum Albumin (BSA)  Incubator at 37oC  Pierce Protein Assay Kit  96-wells flat-bottom plates  Spectrophotometer3.3.2 Recipes  Reagent A, 1L10g 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
  • 20. pH adjusted to 11.25  Reagent B 50ml2gCuSO4.5H2O (4%)Distilled water to 50ml3.4 Procedure3.4.1 Preparation of working reagentTwenty five parts of reagent A, 24 parts of reagent B and one part of reagent C were mixedtogether. The amount of working reagent required for each sample was 1ml for the test tubeprocedure and 150µl for the micro-assay plate procedure. Since the test tube procedure wasalready performed in lieu of the practical, micro-assay procedure was performed. Consequently44 wells of the micro-titer plate were filled with 150µl each working reagent.3.4.2 Preparation of BSA standardThe available concentration of the BSA standard was 2mg/ml in a 1ml volume. This stocksolution was diluted to 80µg/ml to increase the volume to a draw able quantity. A duplicate oftapering concentrations (4-40µg/ml) were prepared with the same diluent (PBS) used to dilutethe sample.3.4.3 Preparation of sampleA two fold serial dilutions of the sample were prepared i.e. 300µl of PBS and 300µl sample andadded 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 12Standard A 40 36 32 28 24 20 16 12 8 4 Blankµg/ml B 40 36 32 28 24 20 16 12 8 4 Blanksample 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 E3.4.4 Micro-plate procedureThe linear working ranges of 4-40µl/ml were used. 150µl of each working standard werepipetted into a micro-well plate. 150µl of the working reagent were added to each well andmixed thoroughly on a plate shaker for 30 seconds. The plate was the covered and incubated at37oC for two hours before cooling to room temperature. The absorbance was then measured at
  • 21. 562 of all the samples on the plate reader and the absorbance value of the blank subtracted fromthe readings of the standards and the unknowns. The blank-corrected 562nm reading for eachstandard vs. its concentration was plotted. The protein concentration of each unknown wasdetermined from the calibration plot.3.5 ResultsAbsorbancesTable 6: Absorbance of both the sample and the BSA standard. 1 2 3 4 5 6 7 8 9 10 11std 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 0.108sample 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 0.114Table 7: Blank-corrected absorbance reading. 1 2 3 4 5 6 7 8 9 10std 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 0.037sample 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 -0.003Table 8: BSA standard and their mean absorbances 1 2 3 4 5 6 7 8 9 10BSA 40 36 32 28 24 20 16 12 8 4stdµg/mlMean 0.3075 0.291 0.2485 0.2175 0.187 0.151 0.1235 0.087 0.056 0.04Ab595
  • 22. Table 9: Sample dilution and mean absorbance 1 2 3 4 5 6 7 8 9 10Sample Conc 1/2 1/4 1/8 1/16 1/32 1/64 1/128 1/256 1/512dilutionMean 1.754 1.08 0.6185 0.3305 0.157 0.07 0.023 0.012 -0.002 -Ab595 0.0015 Absorbance of standard BSA 0.35 0.3 0.25 Absorbance (nm) 0.2 y = 0.0078x - 0.0017 R² = 0.9962 0.15 0.1 0.05 0 0 5 10 15 20 25 30 35 40 45 BSA concentration (µg/ml)Figure 5: Graph showing relationship between Absorbance and BSA standard concentrations for the calculation of sampleconcentration.Protein concentration calculated from the graph e.g. for 1/16 dilutionY= 0.0078x-0.00170.157=0.0078x-0.00170.1587=0.0078xX= 20.35µg/mlConcentration at 1/16 is 20.35µl/ml and therefore the concentration of the undiluted proteinequals 16x 20.35 = 325.6µg/ml
  • 23. 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 theabsorbance of the known and unknown protein concentrations is then done. Ordinarily, BovineSerum Albumin (BSA) is used to make the standard curve. This is probably due to its wideavailability in powder form and can therefore be weighed conveniently in the lab. In addition itdissolves in water to form a colorless solution which reacts with coomassie blue to form a deepblue addition product.Serial dilution of the sample of protein is done because you do not know the concentration of itand it could be less of more than your standard and therefore serial dilution is done to increasethe likelihood that you will be able to produce a sample with absorbance that falls within therange of the standard curve.REFERENCESArber W., Cinn S. (1969) DNA modification and restriction: Annual Review Biochemistry 38:467-500Ausubel, 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: Geneticengineering of E.coli: R-factor DNA proceeding of National Academy of SciencesGriffith, F. (1928) The significance of pneumococcal types. Journal of Hygiene V 27: 113-159Kruger, D.H., Brickle T (1983) Bacteriophage survival; Multiple mechanisms for avoiding theDeoxyribonucleic Acid Restriction Systems of their Host. Microbilogy Review 47: 345-360Mandel M and A. Higa (1970) Calcium Dependent Bacteriophage DNA infection: Journal ofMolecular Biology 53 (1) 159-162Sambrook, J. Russel (2001) Molecular cloning: A Laboratory Manual 3rd ed, Cold SpringHabour Laboratory Press: Cold Spring harbor, NY.Steve Odongo (2011) IPMB General Laboratory ManualWatson, James D. (2007). Recombinant DNA: genes and genomes: a short course. SanFrancisco: W.H. Freeman