How to separate very close protein bands - (Aug/07/2008 )
Dear Friends
I am Sudarshan (Medical university of vienna) want to knoe how to separate two spots which are very close to each other.
i used 12.5% gel ready made from GE helthcare. i found that C-Src protein from santacruz biotechnology giving me two closer bands. i want to separate them.
please i also have another problem is only P-synapsin antibody giving me background problem why?
Sudarshan
Lesser gel percentage will separate your proteins more. Try a 10% gel.
As for the background, it is specific to each antibody. Try diluting more your primary antibody.
lower percentage and longer running time also adds to seperation. But be careful that the gel does not overheat with this additional running time.
i have another one question is what will be the protein concentration when we check phospho proteins
I always use 100ug samples for any phospho antibodies and seem to get pretty good results but have gone as low as 25ug. So depending on which protein u should trouble shoot but should not be very different from regular protein
I am Sudarshan (Medical university of vienna) want to knoe how to separate two spots which are very close to each other.
i used 12.5% gel ready made from GE helthcare. i found that C-Src protein from santacruz biotechnology giving me two closer bands. i want to separate them.
please i also have another problem is only P-synapsin antibody giving me background problem why?
Sudarshan
the question is how near are the bands; we use G11 and L4A1 monoclonal anti-c-Src and get only a single band; if the bands are not very close, it seems to be a problem of the antibody? or are you overexpressing c-Src? in this case, we get also 2 bands for c-Src;
to separate very close bands, better use 2D gel electrophoresis