How much protein needed for sequencing

John E. Fox altabios at bham.ac.uk
Fri Jul 14 06:54:25 EST 1995


In article <3u1o5g$is0 at azure.acsu.buffalo.edu>, xinguo at acsu.buffalo.edu (Xin Guo) says:
>

>
>I have a protein that is transiently expressed in HeLa cells. I want to 
>immunoprecipitate it and then analyse the phosphorylation site. I am


A lot will depend on the background amino acids. We have sequenced
from 800f.mole of protein but that was a very clean sample. A few pico moles
would be OK. The trouble with tryptic digests is that you get a lot of
fragments which can be very hard to resolve. You will have to 
purify your protein before sequencing or you will end up digesting
and sequencing the antibody as well. Maybe a CNBr digest would be 
easier to separate, gives a few large fragments.

John Fox




More information about the Methods mailing list