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

To calculate the volume of the 2.5 M IPTG stock solution needed to achieve a final concentration of 1 mM in a 50 ml culture, you would use the formula C1V1 = C2V2. Here, C1 is the initial concentration (2.5 M), V1 is the volume we want to find, C2 is the final concentration (1 mM or 0.001 M), and V2 is the final volume (50 ml or 0.05 L). Solving for V1 gives V1 = (C2V2) / C1 = (0.001 M * 0.05 L) / 2.5 M = 0.00002 L or 20 μl.

The sample in the lane marked "UN" likely represents the uninduced control. This is included to show the level of protein expression in the absence of IPTG induction.

The samples labeled "P" and "CL" on the SDS-PAGE gel likely represent the pellet and the cleared lysate, respectively, obtained after centrifugation. The pellet contains insoluble proteins and cell debris, while the cleared lysate contains soluble proteins.

Glutathione is typically bound to the resin used in the affinity purification of GST. This is because GST has a high affinity for glutathione, allowing for specific binding and purification of GST from a mixture of proteins.

The sample in the lane marked "W" likely represents the wash fraction. This step is performed to remove any unbound or non-specifically bound proteins from the resin, leaving only the GST bound.

The ligand included in the elution buffer is typically reduced glutathione. It is used because it competes with the GST bound to the resin, causing the GST to elute. This represents a competitive elution strategy.

To calculate the size of the eluted protein, you would compare its migration distance on the gel to that of a set of protein standards of known sizes run on the same gel. This is typically done by plotting the log of the standard protein sizes against their migration distances to create a standard curve, and then using this curve to determine the size of the unknown protein.

The success of the purification of recombinant GST can be assessed by examining the lane marked "E" in Panel C. If a single, clear band of the expected size for GST is visible, and no other bands are present, this would suggest that the purification was successful.

To confirm that the purified protein is GST, you could perform a Western blot using an antibody specific for GST. If the antibody binds to the protein, this would confirm its identity as GST.