The photo shows a certain step of the affinity chromatography purification of the 6xHis-GFP expressed in bacteria.Select the correct statement(s). Question 1Answer1.The picture shows the prepared affinity column.2.The picture shows centrifuged samples.3.The eluted protein collected at the bottom of the tubes (the whitish part below the clear solution).4.In the sample treated with IPTG, the bulk of the the 6xHis tagged protein is found in the supernatant.5.The picture shows the step right before the actual affinity chromatography.

Panel 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

What can be seen in this picture?Question 3AnswerA.the column used in affinity chromatographyB.the bilayer prepared during in vitro translationC.agarose gel prepared for gel electrophoresisD.cuvette with sample, to measure the protein concentrationE.sample prepared for analysis by the Biuret method

Match the stationary phase (Column I) with its corresponding chromatographytechnique (Column II).Column I Column IIP. Protein A 1. Size exclusion chromatographyQ. Sephadex 2. Ion-exchange chromatographyR. Phenylsepharose 3. Affinity chromatographyS. Diethylaminoethyl cellulose 4. Hydrophobic interaction chromatography(A) P-1, Q-4, R-2, S-3(B) P-3, Q-1, R-4, S-2(C) P-3, Q-4, R-2, S-1(D) P-4, Q-1, R-3, S-2

Which tag allows for purification of recombinant fusion proteins using a Nickel affinity column?Give two different elution strategies you could use for the fusion protein referred to in b).Consider the plasmid map for pQE30/31/32 on the next page and answer the questions thatfollow:(2)(1)(2)

The GST-Glutathione is an example of ______ affinity chromatography (a)Antibody-antigen (b)Dye-affinity (c)Enzyme-substrate (d)Metal-affinity