Question 4 purification procedure are shown below. The immunoprecipitation is ca
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Question 4
purification procedure are shown below. The immunoprecipitation is carried out with bind the enzyme of interest. I run an SDS PAGE, and perform a Western blot with the same antibody used for immunoprecipitation, and see one single band at 40kD. However, when I run a native (non-denaturing) PAGE, followed by a Western blot with the same antibody, I see a single band of 160kD. What do you conclude from this experiment? Consider the specific activity table shown in the previous question. In measuring the activity of the enzyme after each purification step, what assumption/s of the Michaelis-Menten equation am I violating? You take total cell lysate, and use immunoprecipitation to purity an enzyme of interest. The purified enzyme has HIGH specific activity, and shows a single band (56kD) on an SDS PAGE. Now, you heat the total cell lysate, and use immunoprecipitation to purify the enzyme of interest. The purified enzyme has LOW specific activity, and shows two bands (56kD and 122kD) on an SDS PAGE. You then heat the total cell lysate, allow it to cool down, and use immunoprecipitation to purify the enzyme of interest. The purified enzyme has HIGH specific activity, and shows one band (56kD) on an SDS PAGE. Finally, you make total cell lysate from a mutant cell. When you use immunoprecipitation to purity the enzyme of interest, the purified enzyme has LOW specific activity, and shows a single band (56kD) on an SDS PAGE. When you heat the cell lysate from the mutant cells and immunoprecipitate the enzyme, the purified enzyme has LOW specific activity, and shows a single band (56kD) on an SDS PAGE. What is the BEST conclusion from this experiment? T F ? The antibody used for immunoprecipitation bound a second protein non-specifically. T F ? The 122 kD protein is a chaperone required for proper folding of the 56 kD protein of interest. T F ? The mutant cells make a non-functional protein that does not refold correctly after heating and cooling. T F ? The 122 kD protein prevents the correct folding of the protein under normal conditions. It is a chaperone for refolding after heating. Two enzymes Xam1 and Xam2 have K_m s of 34mM and 76mM respectively. Which enzyme will have the higher V_max?Explanation / Answer
Please find the answers below:
Answer 2: A denaturing SDS PAGE is run to assess the different isoforms present in a protein. If more than one isoform or physiological entity is present in a protein, it will be resolved on a denaturing PAGE and appear as different bands depending upon their molecular weight. However, a native PAGE is run to assess exactly the same molecular structure of the protein experimentally which is present in the cell itself. Thus, while multiple 40 kDa weight proteins might be seen in a denaturing SDS PAGE followed by investigation using a high-throughput technique such as immunoprecipitation, a single dark band of 160 kDa will be observed in native PAGE.
Answer 3: According to the tabulated data, it can be seen that the sepecific activity of the enzyme is increasing after each extraction step which suggests that purity of the enzyme is increasing after each step, so is its specific activity. However, according to Michaeli's assumptions, the native biological conditions of the enzyme is changing everytime an extraction process or enrichment step is followed over here.
Answer 4:
Choice 1 is false: The data shows that the immunoprecipitation results show differential activities even after heating the cell lysate. This could not have been possible if the antibody was not specific enough.
Choice 2 is true: According to the data, heating the sample results in formation of two bands which clearly shows that the 122 kDa protein is a chaperone by nature.
Chocie 3 is true : According to the data, the mutant cells produce only a single band of 56 kDa on SDS PAGE suggesting that they do not produce the chaperone protein.
Choice 4 is true: The heating and non-heating steps involve expression of 56 kDa and the both 56 and 122 kDa proteins which clearly suggest the differential role of chaperone protein on protein folding depending upon the ambient temperature.
Answer 5: According to the Michaeli's-Menten equation, the Vmax and Km are inversly proportional to each other. Thus, the enzyme having low Km will have high Vmax and vice versa. Thus, the enzyme Xam1 will have higher Vmax.
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