4. You take total cell lysate, and use immunoprecipitation to purify an enzyme o

Question

4. You take total cell lysate, and use immunoprecipitation to purify 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 purify 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? OT OF O? The antibody used for immunoprecipitation bound a second protein non-specifically. DI OF 0 ? The 122 km protein is a chaperone required for proper folding of the 56 kb protein of interest. The mutant cells make a non-functional protein that does not refold correctly after heating and cooling. DI DE O ? The 122 kD protein prevents the correct folding of the protein under normal conditions. It is a chaperone for refolding after heating.

Explanation / Answer

Ans-1 true, it might be possible that some other protein may be present having similar domains like the desired protein or some other factors make the antibody to bind another protein.

Ans-2 false because in first case there was no chaperone present.

Ans-3 true it might be another protein which is non functional due to absence of some required factor.

Ans-4 true, the above factors shows the same manner of 122 kd protein.

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