I am trying to do a Co-IP, I think that my interactions is very weak and/or transient, because I can't see anything by regular Co-IP.
Anyone use or know a protocol for Co-IP using crosslinking?
You could try the method used by Kane and colleagues years ago when looking at V-ATPase complexes in yeast. See Kane, PM (1995) JBC 270:17025-32, PMID: 7622524. She treated extracts with a reversible crosslinker, DSP, before the IP step.
Pierce have some good resources for cross-linking. Lots of info here: http://www.piercenet.com/browse.cfm?fldID=020306
I did some crosslinking for IP a long time ago, and it was tricky to work out which crosslinker was appropriate. I found the Pierce resources very helpful.
The procedure to cross-link between protein-protein and protein-DNA using formaldehyde are widely used in Chromatin-immunoprecipitation. The procedures are available established and online from Upstate, NEBioLab, Promega, Clontech, Pierce, QiaGen or many other relevant publications.
The reversal of cross-linking is performed by boiling in the presence of SDS. Analysis by PAGE and Western-blotting. I have used the identification of minute quantities of IP proteins using this procedures. Please refer to my recent paper in J.Biol.Chem. 2010.
If you use a 35S-methionine labelled protein you can visualize by autoradiography and you will eliminate the disturbance of IgGs used in the IP in your Western Blots.
Best of luck.
an alternative to cross-linking is to covalently couple the Abs directly to the bead surface using epoxy or tosyl chemistry.
Nucleic Acids Research, 2014, Vol. 42, No. 14 9021
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