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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

-sudarshanspatil-

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.

-Madrius-

lower percentage and longer running time also adds to seperation. But be careful that the gel does not overheat with this additional running time.

-WeStErNbLoT101-

i have another one question is what will be the protein concentration when we check phospho proteins

-sudarshanspatil-

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

-WeStErNbLoT101-

QUOTE (sudarshanspatil @ Aug 7 2008, 09:13 AM)
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


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

-The Bearer-