Non reducing Co-IP and 2 Ab that detect light chain only - Co-IP help! (Aug/29/2006 )
Can anyone offer any advice? Its that same old problem. Im trying to do a Co-IP where one of my proteins runs at the same position as the heavy chain. I have tried lysing my cells with RIPA buffer and resuspending in non-reducing buffer (Pierce) [100oC for 3min before loading] but Im still getting heavy and light chains. Im running the samples on nupage bis-tris gels with MOPs buffer (both Invitrogen), could this be effecting the samples?
Also I have read in another forum that it is possible to get a secondary Ab that only picks up the light chain which would simplfy things alot! Has anyone heard of this and can anyone recommend one?
Would appreciate any help - many thanks!
Not sure about the ab to detect light chain only. Could you cross-link your ab directly to the protein G sepharose to avoid this problem? Or maybe use a different species detection ab to the one you used for the IP?
There are no other antibodies available for my proteins which leaves me restricted in that way.
I havent tried cross linking, to be honest I was trying to avoid complicating the IP as it itself works so well for all the other detections. maybe i'll just have to resign to cross linking!
Otherwise I'm looking at getting a kit that removes heavy and light chain. Any idea which is better -trueblot or the pierce kits or santacruz kits?
Sorry, Keeva. I haven't used either of those kits.
My old boss used to regularly cross-linked abs to protein G-sepharose. The abs are bound to the protein G sepharose overnight at 4degrees on a rotater (100µg ab to 1ml of beads). Then cross-link them with freshly made 40mM dimethylpimelimidate. 2HCl (from Sigma) in 0.1M Borate pH9 for 45mins at room temperature on a rotater. Then wash twice with 50mM ethanolamine pH 8 (incubate the 2nd wash for over 30mins on the rotater) and wash twice in 50mM Tris Cl pH7.5.
Good luck with whatever you plan to use.
I'll maybe try cross linking then