I would like to run a GFP quimeric protein and determine its molecular weight. I need to detect the GFP protein trought Fluorescence.
Therefore I can´t just run a regular native gel. Does anybody nows how much SDS I can add to the SDS-PAGE without denaturing the GFP part of the protein?
Hi maximilian thanks for your answer
Have you used 0,1% SDS in all places (Runing buffer, gel and sample buffer?). Have you checked that proteins run acoording to its MW with that amount of SDS?
I am not sure this will work with your particular protein, but the basis of a far western is to run your proteins out then blot them to nitrocellulose and refold them on the nitrocellulose in the presence of BSA. This technique may work for your protein. I have attached a virus overlay protocol that we use, but you would only be using the first 2 steps. Could you not just use an anti-GFP antibody and a molecular weight blot marker like MagicMark XP from life tech? or do you need it on a gel for some reason?
Cheers
Ron
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