Dear Hila,

You could try to use substrate trapping mutants – if that fits your purpose. There would be at least 3 ways of doing so: (1) You could produce a phospho-mimic of the PP2A substrate (has worked, for example in Arnold & Sears MCB, 2006 for c-Myc:PP2A). (2) You could mutate the active site of PP2A. This has worked for a number of other phosphatases, however has not yet been reported for PP2A – so you’d be the first ☺. (3) if you have a good structural knowledge about the interaction, then you could introduce a cysteine in your kinase in a way that this new cysteine will form a disulphide bond with the PP2A cysteine 269 (see PDB 4I5L, for example). Good luck.

Hi,

perhaps you could try to perform a reversible crosslinking with formaldehyde for your Co-IP. See this for more details:

http://www.piercenet.com/guide/in-vivo-crosslinking

Best,

Lukasz

Thank you for your help. I'll look into your ideas.

The most straightforward approach would be to use a reversible cross-linker. This would also be easier, but less elegant than Stefan Arold's  suggestion. There are many different cross-linkers, and Pierce has a very good selection  with detailed information.

Thank you all!!!!

To stabilize the weak interaction between Hras and Gal1 we used BiFC.

http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0011991

Good luck.

Just out of curiosity....how about trying a simple method like yeast two hybrid to first confirm the interaction strength.....some of my colleague say that a very strong two hybrid interactions help them get a good invitro pull-down....weaker interactors do not perform well in pull down.....just a piece of information....may be of some use...

thanks, Y2H is in me list.