My antibody is forming a precipitate! - Why? (Jan/22/2007 )

Hi All,

When I elute my antibody (on protein G column) using glycine buffer at pH 2.7 a precipitate appears to form in the eluate. Why is this happening? Is it because it is such high concentration? Is it bad in any way? It doesn't happen to other antibodies I purify, just this one particular cell line. Is it OK to filter this through a 0.2um filter or could this lose the antibody? Will the precipiate remain if I dialyse it against PBS?

If you could answer any or all of these questions I would be most appreciative!!

Thanks

-MABgirl-

I also have the same problem with one antibody.
I would like to know the reason for it and how to solve it.

-Minnie Mouse-

QUOTE (Minnie Mouse @ Jan 23 2007, 11:38 AM)

I also have the same problem with one antibody.
I would like to know the reason for it and how to solve it.

Minnie- Did it affect the antibody function?

-MABgirl-

do you add concentrated buffer to the fraction to raise the pH immediately after elution?

you could be denaturing your antibodies with the low pH.

-mdfenko-

yes i add 1M Tris/HCl to neutralise my antibody immediately after elution

-MABgirl-

Hi mdfenko,

Is it better to neutralize the antibody with 0.5M Tris or 0.1M Tris?

Thanks in advance.

-Minnie Mouse-

QUOTE (Minnie Mouse @ Jan 23 2007, 05:45 PM)

Hi mdfenko,

Is it better to neutralize the antibody with 0.5M Tris or 0.1M Tris?

Thanks in advance.

i neutralize with 1/5 of the fraction volume 1 M phosphate pH 8.0. i put it into the tube and elute directly into the neutralizing buffer, mixing as soon as the fraction is finished.

-mdfenko-

QUOTE (mdfenko @ Jan 24 2007, 12:24 PM)

i neutralize with 1/5 of the fraction volume 1 M phosphate pH 8.0. i put it into the tube and elute directly into the neutralizing buffer, mixing as soon as the fraction is finished.

Thank you mdfenko

-Minnie Mouse-