Hi, I need help understanding what could be the issue with my endogenous Co-IP's. The TCL shows I have enough amounts of the proteins but my IP samples hardly show any signals and most of the times if there is a signal it shows up in the control IgG lanes as well.
To give you an idea about my work flow - I work with B-cells and I am trying to see the Co-IP of a membrane protein with another protein. For the lysis I use - 0,5% NP 40 buffer with 50mM tris-cl (pH 7.4 - 7.6 - I measure and adjust the pH when the buffer is cold at 4deg), 150mM NaCl, 1 mM EDTA. Just prior to starting the IP I add the Protease Inhibitor cocktail, 1mM DTT and 1mM Na3V04 ( I wish to look at phospho Tyr signals). I usually incubate my cells with the LB for 15-20 mins and I have even tried passing them through an 18G needle to get a better yield of the protein ( this is worked sometimes but lately again its a problem). Once I have the lysate I measure the protein conc and incubate 500microgm of total protein with 1 - 1,5 microgm of antibody overnight. Next day, I wash magnetic beads and incubate them with this lysate for 2-3 hrs. Wash this mix 3-4 times and add 2x laemmli buffer. I heat the samples at >90degrees for 5-7mins and load the samples to proceed with the WB.
The same protocol works when I work with over expressed proteins and I see the Co-IP very well.
I would appreciate if you can suggest what else could I try to make my endogenous IP work. I am really at my wits end. I have checked the expression of my proteins of interest by a simple cell lysis and both the proteins are expressed abundantly. Also how common is it to pre-clear the lysates with the control IgG/beads and then proceed to incubating with the IP antibody.
These are some possibilities you might want to consider.
1. Due to overnight incubation your protein might be degraded (may not be fully degraded when over expressed). Check your protein lysate after IP for expression of target protein.
2. Try Antibody+beads first and then add lysates and try shorter time such as 2-3 hours in 4 degree C with rocking.
3. Add 1mM PMSF (stock 200mM in ethanol) to lysis buffer to minimize protein degradation.
4. I would recommend to omit DTT if possible. It can be added in sample loading buffer. DTT might be effective when the protein conc is low.
5. You may also add NaF to preserve phosphoproteins in addition to Na3VO4
Hey ! Thank you so much. I think all these suggestions are great. Will try them soon ! I had a feeling that during the the ON incubation the protein is getting degraded but now that you mention it as well, I think thats exactly whats happening.
I agree, I'd omit the reductant from your IP and shorten your incubation...my suspicion is that it's an issue of diminishing returns with 90% of the interaction occurring in the first few hours. I also worry about what seem to be indiscriminate or contaminating bands on your WB. Perhaps an elution off your beads could be less stringent than a full boil (ie. a strip solution) or better yet peptide or cleavage based.
Hello ! thank you for your suggestions. Once I repeat the IP with your suggestions I will update you guys !
Hi Just wanted to thank you guys once more :) It worked ! I could see a good pulldown. I am on my way with the repeat ! keeping fingers crossed !
Hi Zankuti, I have then same issue with endogenous protein immunoprecipitation. Could I known which steps above solved your problem? Is overnight too long?
Another thing is you works with B cells, do endogenous Igs matter? Do you incubate antibody with lysates first or antibody with beads first? Thanks.
Hi Yuwei, I eliminated the DTT and now for endogenous IP's I usually incubate the antibody with magnetic beads for 15minutes
and then add these to the lysates for 3-4 hours. And no the endogenous Ig's don't interfere.
Zankruti Dave
Hi Zankruti, thank you so much for your help, I had the same problem as you but i just want to know did you exclude DTT totally from your procedure? ... did you replace it with other reagent? ,, usually I use DTT in sample buffer then put it at 98 degree for 5 minutes before loading in SDS -page, so in your case, you load the samples directly after 98 degree with loading sample buffer without DTT? .. could you please clarify this point for me?.. thanks in advance
- Badges
- Science topic
- Similar topics
- Biological Science
- Biochemistry
- Enzymology
- Enzymes