Describe and explain fully what you observe in Lane 5 of the agarose gel. Why is this samplenecessary?Determine the size of the linear bands in Lane 6 -8 using the graph paper provided. You mustshow all your calculations and hand in the graph generated.Do the bands in Lanes 6-8 of Figure 1B correspond with what you would expect for theserestriction digestions of pGEX4T-1? Explain your answer in full, with reference to the plasmidmap in Figure 1A and the agarose gel in Figure 1B and show all your calculations (you mustcompare your predicted band sizes to the calculated sizes from question 2i above).(3)(10)(8)40a)b)c)d)e)f)g)h)i)Question 3Panel C of Figure 1 on page 4 shows the results of an induction study and affinity purificationof the 26 kDa Schistosoma japonicum GST protein. The protein was induced by the addition ofIPTG to a final concentration of 1 mM and then purified using affinity chromatography. Answerthe following questions based on Figure 1, Panel C.If a stock solution of 2.5 M IPTG was used for induction, calculate the volume of stock solution(in μl) that would be added to a 50 ml culture to obtain the required final concentration. Youmust show all your calculations.What is the purpose of including the sample in the lane marked “UN”?What is represented by the samples labeled “P” and “CL” on the SDS-PAGE gel? Your answermust refer to the different fractions obtained after centrifugation and what is contained in eachof these fractions.Which molecule was bound to the resin used in the affinity purification of GST? Why is thisparticular molecule used to purify GST?Briefly describe the experimental procedure carried out to generate the sample shown in the lanedenoted “W” and explain why you would perform this step.What ligand was included in the elution buffer when you carried out this experiment, why wasit used and what kind of elution strategy does this represent?Explain how you would calculate the size of the eluted protein, with reference to one of the otherlanes on the gel. NOTE: you do not need to generate a graph or calculate the size, only explainHOW you would do this.Describe fully whether you think that the purification of recombinant GST was successful. Inyour answer, refer to the lane denoted “E” in Panel C.What additional experiment could you conduct to confirm that the purified protein is, in fact,GST?(3)(2)(4)(3)(3)(3)(3)(3)(1)25
Question
Describe and explain fully what you observe in Lane 5 of the agarose gel. Why is this samplenecessary?Determine the size of the linear bands in Lane 6 -8 using the graph paper provided. You mustshow all your calculations and hand in the graph generated.Do the bands in Lanes 6-8 of Figure 1B correspond with what you would expect for theserestriction digestions of pGEX4T-1? Explain your answer in full, with reference to the plasmidmap in Figure 1A and the agarose gel in Figure 1B and show all your calculations (you mustcompare your predicted band sizes to the calculated sizes from question 2i above).(3)(10)(8)40a)b)c)d)e)f)g)h)i)Question 3Panel C of Figure 1 on page 4 shows the results of an induction study and affinity purificationof the 26 kDa Schistosoma japonicum GST protein. The protein was induced by the addition ofIPTG to a final concentration of 1 mM and then purified using affinity chromatography. Answerthe following questions based on Figure 1, Panel C.If a stock solution of 2.5 M IPTG was used for induction, calculate the volume of stock solution(in μl) that would be added to a 50 ml culture to obtain the required final concentration. Youmust show all your calculations.What is the purpose of including the sample in the lane marked “UN”?What is represented by the samples labeled “P” and “CL” on the SDS-PAGE gel? Your answermust refer to the different fractions obtained after centrifugation and what is contained in eachof these fractions.Which molecule was bound to the resin used in the affinity purification of GST? Why is thisparticular molecule used to purify GST?Briefly describe the experimental procedure carried out to generate the sample shown in the lanedenoted “W” and explain why you would perform this step.What ligand was included in the elution buffer when you carried out this experiment, why wasit used and what kind of elution strategy does this represent?Explain how you would calculate the size of the eluted protein, with reference to one of the otherlanes on the gel. NOTE: you do not need to generate a graph or calculate the size, only explainHOW you would do this.Describe fully whether you think that the purification of recombinant GST was successful. Inyour answer, refer to the lane denoted “E” in Panel C.What additional experiment could you conduct to confirm that the purified protein is, in fact,GST?(3)(2)(4)(3)(3)(3)(3)(3)(1)25
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