Hi,

I'm performing IP with Dynabeads Protein G. I'm trying to purify a protein with a V5 tag in it native state.

I've already tried elution in glicyne pH 2.7 (according to the recomended protocol) and nothing.

Anyone have a efficient protocol to elute proteins from dynabeads in its native state?

Thanks in advanced!

Hi,
I am trying to analyse interactors of a Flag-taggeted protein using Flag-M2 agarose for IP. Expression of Flag constract is inducible by doxocyclin. For Flag-IP I take dox+ and as a control dox- cell extracts. The problem is that I got no difference between control and dox+ samples in a cell fraction where the concentration of the interactors is high. Any suggestion?
Thanks

Hi,

I'm trying to precipitate my protein of interest from total cell lysate, along with interacting proteins, so that they are visible on silver stained gels. The problem is that I am getting a lot of nonspecific bands sticking to control IPs (beads alone or with nonrelated antibody).

I have tried preclearing, and using higher ultracentrifugation, which have improved the stain a bit, but still too many nonspecific bands. Any suggestions? I'm about to try washing with different salt concentrations (usually use lysis buffer with 1% triton and 150mM NaCl).

Hi,

Does anybody know a commercially available antibody against progesterone receptor (PR) that can be used for IP of both PR-A AND PR-B?