I am looking for efficient kits for Co-IP using mouse and rabbit IgGs. Effectively co-IP output should be minimally contaminated with IgG heavy/light chains. Any idea?
Hi George, if you don't want your eluted IPs to be have any Abs, you will have to attach your Abs directly to an activated resin (CNBr Sepharose, Affigel, etc.) .
If you use protein A/G resins to attach your Abs, most likely the antibodies will come off the resins in your elution steps.
Thermoscientific Pierce has a Crosslinking CoIP kit. Ideally the crosslinked antibodies shouldn't come off during elution.
Thanks a lot for all the input. Actually I have already used the crosslinking coip kit from Pierce, but still I see heavy contamination with IgG chains. I followed the protocol precisely and I just played with amount of antibody. I will revisit the procedure and see what was wrong
Hi Steingrimur. After extensive washing with buffer from kit I normally boil beads with Laemmli sample buffer (for routine work; custom made) but I also dissociate immune complexes with acidic 0.2 M glycine (also custom made) but in every case I see huge heavy and light chain IgG bands. I know that I must be doing something wrong, but I have no idea what. I can return to Protein G beads but proteins we need to pull down are in similar MW range (25-50 kDa) with antibody chains
Hi George if you are using the coip kit from Pierce, you could try loading up the resin with your Ab, do the crosslinking with the kit and afterwards treat it with the glycine buffer to remove any uncrosslinked Abs. before you apply your sample to the resin and do the IP.
Just to note, the DSS crosslinker in this kit is very labile. You have to use it within minutes of dissolving it. If you try to store it in a solvent that has any H2O in it, it will degrade really quickly and become useless.
Hi Steingrimur and apologies for late response. These are very helpful hints. Thanks a lot. I am going to try them. Do you think it might be possible to substitute crosslinker with some alternative?
Hi George, no prob.
I've attached some publications on crosslinking Abs to protein A resin:
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2643637/
http://www.acsu.buffalo.edu/~lread/pdf/2001molbiochemparasitol.pdf
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2773615/
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1133783/
The main thing from these publications is that many of them use dimethylsuberimidate as a crosslinking agent, and that they extensively wash the antibody resin before using it.
Hope this helps you.
Hi,
Dynabeads Antibody Coupling Kit (143.11)
Dynabeads and co-IP - 143.21D
These kits were used in the following references:
Nucleic Acids Research, 2014, Vol. 42, No. 14 9021
Methods and Protocols, Methods in Molecular Biology, vol. 1102, 2014
PLOS ONE | March 2014 | Volume 9 | Issue 3 |
PLOS ONE | January 2014 | Volume 9 | Issue 1 |
kind regards
ketil
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