ian isn't there a need to block the beads
with something that won't interfere withthe
protein-A?
"Ian A. York" wrote:
> 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
