I have been doing co-immunoprecipitations for a while now using my own buffers (CHAPS lysis buffer + protease & phosphatase inhibitors) and Dynabeads protein G beads. Useful information:
-The antibody I use for pulling down protein X we have produced in rabbit and shows specificity on WB (this is serum, so I do not have an exact concentration of the antibody, there is no other known working antibody for this protein).
-I use cortical tissue from P14 mouse pups and for each sample: 500ul lysate tissue (185mg/ml protein) - 25ul antibody - 50ul Dynabeads
-The antibody heavy chain smear overlaps with the co-IPed protein Y I'm trying to visualise (also with rabbit antibody). So I have been cross linking the serum-antibody (protocol by Thermo Scientific Pierce) to the beads by use of BS3, to prevent the smears. I've got 50mg of BS3 stocked and I weigh a certain amount every time I need it (I always make it fresh)
-I cannot heat the sample during elution because then I cannot visualise protein Y on WB, so I use 50mM Glycine, pH2.8 for 2' @ RT and after add 2M Tris, pH 9.4
-The first time I did the experiment I could see proteins X and Y after the co-IP (cross linking worked), unfortunately I did not include a control.
So, I have been trying to reproduce the experiment with an additional control antibody (rabbit IgG), but now in the sample where I use the antibody-serum for protein X, I cannot see protein X after the co-IP on WB.
--> In both antibody-serum and control samples: on WB it shows proteins X and Y in the input control, but in the co-IP sample its completely blank. No smears either (cross-linking the antibody to the beads works apparently).
I cannot think of what has changed after the first experiment and why I don't get results anymore after trying several times. Im afraid something has happened during the cross linking/antibody incubation.
Sorry to overload with all this information, but I'd appreciate any thoughts/suggestions on this.
Dear Amila,
I do not have a suggestion for the cross linking issue, but you might also consider using a different secondary antibody or IP method called trueblot (http://www.rockland-inc.com/TrueBlot-IP-Western-Blotting-Main.aspx). This will prevent detection of you antibody smears via a different approach. Hope it helps.
Wim
Hi Amila,
try to increase the amount of protein, try to go up to 500mg at least.
Co-IP from tissues are very hard to get compared to overexpression experiments since steady state levels of your protein of interest might be too low.
Good Luck!
Cross-linking the antibody to the resin can kill the antibody.
However, I would suggest you to perform the elution with 2x beads volume of 100mM glycine (pH 2.2), 5 mins at RT. Repeat the elution twice and rebuffer with tris pH >8.8. take the beads, add Leammli buffer and cook them. In WB analysis, you should load parallely the eluates and the beads to be sure in which fraction your proteins X&Y are enriched.
Good luck
Thank you all, next month I will do more co-IP experiments and I will let you know the results!
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