In article <20010315191005.12388.qmail at ww02.jatek.com>,
Manjili <mmanjili at yahoo.com> wrote:
>>buffer or PBS. I incubate antibody-antigen at 4 degree for 2 h and then add
>protein A sepharose to the mixture followed by incubation at 4 degree
>overnight while rotating.
Overnight is way longer than necessary, and can definitely cause
non-specific binding. Try adding antibody for 1 hour on ice, and then
protein A-sepharose for 90 minutes while rotating.
Also consider the possibility that it's not actually binding, but proteins
coming out of solution (or not being cleared properly): if the protein
precipitates, then it'll come along with your protein A-sepharose pellet.
Spin the lysate aggressively before adding antibody. Or, if you don't
mind spending a bit of money, try Spin-X microfuge tubes or something
similar: a .45 micron filter clears the lysate out very nicely.
Ian
--
Ian York (iayork at panix.com) <http://www.panix.com/~iayork/>
"-but as he was a York, I am rather inclined to suppose him a
very respectable Man." -Jane Austen, The History of England
