Gwendolyn Quintana 01187235 04 11 13 Cellular Biology Lab Homework 3 You may use the lab manual pre lab lectures and the Internet to help you answer the questions below You may not discuss this homework with your classmates Plagiarism will result in an automatic zero Part A 1 In the cell bio lab we use SDS PAGE gels purchased from a company however you can make you own polyacrylamide gels List all of the ingredients found in an SDS PAGE gel Which ingredients are responsible for polymerizing the solution How does the percentage of acrylamide effect the migration of proteins ex 4 gel vs 18 gel Ingredients included in SDS PAGE gel are agarose or acrylamide with pH specific buffer in water bisacrylamide and sodium dodecyl sulfate Additionally ammonium persulfate and TEMED free radical stabilizer are added to allow polymerization of the gel The stacking gel 4 is responsible for allowing the loaded samples to align simultaneously at the bottom of the stacking gel where to beginning of the resolving gel 12 begins The percentage of acrylamide in the gel allows the proteins to migrate faster when at lower percentages 4 than at higher percentages 12 and vice versa Likewise at higher concentration of acrylamide proteins are allowed to travel more uniformly and are less likely to result in additional effects such as smiling or smearing of the protein Part B You are asked to run an SDS PAGE analysis of samples from an affinity chromatography experiment performed by your TA A recombinant protein with a hexahistidine tag was captured in a nickel agarose bead matrix and eluted off the column with 75mM imidazole Your TA was hoping to make about 2 mg mL of the protein You are given the following samples to prepare and load into an SDS PAGE gel 10 uL 75 mM Imidazole 10 uL pure recombinant protein at 2 mg mL 10 uL recombinant protein in 75 mM imidazole elution fraction 1 Describe how you would prepare the three samples to load into the gel Of course all samples would be prepared the same as to set a control Assuming all tubes have been aliquoted to eppendorf tubes all tubes are added 5uL of SDS sample buffer Samples are then to be put in the dry bathe at 100 C for 5 minutes If possible place the samples on ice A molecular weight marker would need to be obtained to load into the gel as well 2 Label the gel lanes with the appropriate contents The recombinant protein should exhibit one band at 66 000 Daltons Lane One molecular weight ladder Lane Two elution fraction Lane Three pure recombinant protein Lane Four 75 mM Imidazole 3 Compare the elution lane to the positive control lane How do you know the recombinant protein is present in the elution sample How pure is the elution sample compared to the control The elution lane has a band at 66 000 Daltons as does the positive control the positive control has no band representative of the Imidazole where as the elution sample does Another important difference between these two wells is that the positive control has a visibly thicker band at 66 000 Daltons than does the elution sample Because the experimental elution sample shows a band where positive pure recombinant protein sample is shown we know that there is pure recombinant protein present in the experimental sample The only contamination visible can be attributed to the Imidazole in the elution sample The elution sample purification other than what previously stated is equally pure as the positive control if not more pure because the band size is thinner in the elution sample than that of the positive control Nonetheless this could simply be due to the fact that there is just a higher concentration of protein than that of the elution sample Hence the elution sample and positive control are equally pure except for the presence of Imidazole in the elution sample 4 What assumption can you make about the amount of recombinant protein in the elution sample What assay can you perform to find the accurate concentration of the elution sample We can assume that there is recombinant protein represented at 66 000 Daltons in the elution sample We cannot assume the exact concentration of recombinant protein in the elution sample by SDS PAGE Assuming that we have remaining sample of the initial sample we can do a Bradford protein assay to measure accurate concentration of the elution sample 1 2 3 4 1 7 0 8 0 5 8 4 6 Protein marker values are in kDa units 2 5 Part C To determine the concentration of the purified recombinant protein you decide to perform a Bradford Assay You make a set of BSA standards and made a 1 5 dilution of the recombinant protein sample All samples were measured with the spectrophotometer at 545nm If the absorbance of your TA s sample was 0 63 units what is the concentration of your TA s purified protein sample The recombinant protein concentration of TA s 1 5 dilution sample is about 282ug mL The TA s original sample is about 1410ug mL protein concentration f x 0 x 0 55 R 0 93 Abs 595 nm 1 2 1 0 8 0 6 0 4 0 2 0 0 500 1000 1500 2000 2500 Concentration ug mL