Identification of monomers/dimers in whole cell lysate via SDS-PAGE - (Nov/25/2007 )
I was hoping someone can help me out here.
I am planning to perform SDS-PAGE against whole cell lysates (mammalian cels) in order to determine my target protein.
Unfortunately, there is one thing that i need to clear before i can perform the experiment.
The protein that i am working with forms monomers within the cytoplasm but upon ligand binding, it dimerizes and is translocated into the nucleus.
By just treating my cells with the SDS loading buffer, would it be sufficient to lyse both the cell membrane and the nucleus membrane, thus allowing proteins from both the nucleus and cytoplasm to be analyzed? or would it be necessary for me to extract the nucleus from the cytoplasm fraction and analyze the fractions separately?
Thank you in advance.
you are right...SDS will take care of lysing both membranes...you can stick on to that..good luck
better is to subfractionize the cell in membrane fraction and nucleus;
of whole lysate you cannot assign the polypeptides to the subfraction;
are you sure that after separation by SDS-PAGE dimers are still dimerized? better to use native PAGE if it is not the case...
Thanks very much.
I am not really interested in subfractioning my samples cause i already know where they localize within the cell before and after dimerization.
i was just curious whether SDS would take care and lyse both the cellular and nucleic membrane.
In the case of dimerization, i am not very sure.
I have read in the topics within the forum regarding native-PAGE but i understand that its difficult to actually tell the real size of my target protein.
Although i mentioned that the protein exists as a monomer prior to dimerization, but as a monomer, it binds to other proteins (to form a complex) to maintain their stability within the cytoplasm.
I don't really know the size of the monomer complex, so i can't really tell whether its my target protein or not.
That is why i though that SDS-PAGE would be the most effective way to determine my protein dimerization.
To look at dimerization via SDS-PAGE, I came up with some thoughts including:
1. Removing the sulfide bond treatment agents such as BME or DTT within my sample buffer.
2. Not to boil my samples as not to break the Van-der Waals bonds among the dimers.
I've actually not tried them out yet but hope to get some results out soon.
Any additional suggestions would help.
Anyway, thanks again for the advice.
I'll try working it out and see whether i get any promising results.