The results from an enzyme purification procedure are shown below. The immunopre

Question

The results from an enzyme purification procedure are shown below. The immunoprecipitation is carried out with an antibody known to bind the enzyme of interest. Step Total cell lysate Salt precipitation Immunoprecipitation Specific activity 52 U/mg 450 U/mg 20000 U/mg I run an SDS PAGE, and perform a Western blot with the same antibody used for immunoprecipitation, and see one single band at 75kD. However, when I run a native (non denaturing) PAGE, followed by a western blot with the same antibody, I see a single band of 150kD. 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?

Explanation / Answer

Answer:

1)

Native gels doesnot contain denaturing agent (SDS or a reducing agent), electrophoresis buffer doesnot contain SDS and samples are also not heated. The MW of intact antibody (example IgG) is 25*2 + 50*2 = 150 kDa (two light chains and two heavy chains)

Under reducing conditions, the disulfide bonds that joins the light chains together and the heavy chains together are broken, this results in the formation of single band at 75 kDa (due to one light chain and one heavy chain) 25 kDa + 50 kDa = 75 kDa.

2) Assumption of Michaelis-Menten equation that is violated is:

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