[Nescafe]

Hi,

I know that EMSA is used to study protein:DNA or protein:RNA binding where as western blot is used to detect protein. What I really don't get is why you cant use each of these techniques for both purposes. For instance why can't you just run an EMSA and try to find the protein you are looking for. Lets say you wanted to see the level of phosphorylated protein X, why is it that western blot would be used to determine it rather than EMSA.

Let me elaborate, lets say the protein of interest gets phosphorylated (PROTEIN A) and binds to another protein (PROTEIN B) and together they bind to DNA. If you want to find the phosphorylated levels of Protein A and B alone you do Western blot where as if you wanted to study ProteinA:ProteinB- DNA complex you use EMSA, can anyone help me get out of this tangled thought.

Cheers,

Nescafe.

[rjb]

Nescafe,

I might be wrong, but I suspect this is due to the fact that most Western blots processes are not just about detection, but also separation. To properly separate proteins of similar size, it is generally necessary to carry out the process under denaturing conditions (SDS). These conditions would be inappropriate for EMSA where the binding can presumably only occur with undenatured native structure protein... I also suspect that buffer requirement are quite different between the two processes...

Hope that helps.

Kind Regards

R

[Nescafe]

Nescafe,

I might be wrong, but I suspect this is due to the fact that most Western blots processes are not just about detection, but also separation. To properly separate proteins of similar size, it is generally necessary to carry out the process under denaturing conditions (SDS). These conditions would be inappropriate for EMSA where the binding can presumably only occur with undenatured native structure protein... I also suspect that buffer requirement are quite different between the two processes...

Hope that helps.

Kind Regards

R

If what you say is true then it makes sense. Thanks. If noone else rights anything to oppose it then I'll believe you to be correct.

Nescafé