immunoprecipitation problem - (Oct/11/2007 )

hello everyone,
i have a serious problem with an immunoprecipitation of certain protein,
actually i ve been trying to immunoprecipitate this protein with no success, searching the problem, i found that when using the tampon of lyse of the ip (NP40 1%) with the tampon of laemmli (added before the migration) i have a high background on my western blot (which i dont get when using the tampon of laemmli alone) i'm suggesting that the tampon NP40 1% is not suitable for this protein and the protein is degradated, i ve already used the same composition to immunoprecipitate another protein of the same family and that worked.. so im not sure what to do.. can i do the immunoprecipitation in the tampon of laemmli with the B-mercaptoethanol and anti-protease!! that can be stupid but im looking desperately for any possible solution,
any suggestion are welcomed,
have a nice day,

---

Are u sure that your antibody works for I.P????????

---

np-40, like triton x-100, can displace sds from the protein. what you may be seeing in the blot is a smear caused by varying degrees of sds incorporation with your protein.

---

thanx so much for your rapid answers, it's really urgent for me,
well first, for the antibody im using; yes it can be used in the ip
secondly: in the habitual buffer of lyse im using for the WB : i have laemmli sample buffer (biorad containing SDS as described in the info sheet), with this buffer i see just one band, no background..
the problem occured when using the buffer of lyse for the ip, it contains:
tris base pH8 20mM
NaCl 140mM
Np40 1%
PMSF, antiphosphatase et anti protease
i thought that the problem was of the buffer because running a sample lysed with this buffer (+ same volume laemmli added just before the migration) gave a background and didnt give the same blot of a sample lysed with just laemmli,
is anyone have ever tried to do an ip with the buffer of laemmli (with the bleu bromophenol in)
again thanx so much for ur help
have a nice day

---

We sometimes do IP after lyzing cells or elute protein using laemmli buffer, but we don't include bromophenol blue in the buffer. In that case, we dilute the sample (to reduce the concentration of SDS) before do IP. Usually we try to dilute so that final conc. of SDS is about 0.1-0.5%. It works if you just want to pull down the protein (if your ab still can recognize the epitope). If you want to study protein interaction, though, it really depends on the nature of your protein and its interacting partners, although it is quite hard to maintain interaction in the high amount of SDS that laemmli buffer contains.
If you think that the smear is due to your lysis buffer, you can try using other lysis buffer, I am sure the papers and/or the protocol sites will have plenty of lysis buffer for IP. If your protein is cytosolic, you don't even need detergent. Or else, you can try different detergent, e.g. 1% Triton X-100, or 1% DOC, or 0.1-1%SDS, or some combinations between them. It depends a lot on your protein, so you have to try out.

---

Thanx so much,
i guess i should try an ip with one of these conditions, but i guess u r right, i won't do the test with laemmli biorad (it contains already bleu bromophenol and 2% SDS) i guess it doesnt worth the try
i will try it soon and come back if i get the same problem
thanx again for everybody
regards

---

Hello,

I was also seeing smears on Western blot when I boiled protein A Sepharose beads in loading buffer, spinned them down and loaded the supernatant. Looking very carefully under a lamp I saw that the beads somehow desintegrate (?) after heating, and even if I centrifuged max speed for a couple of minutes to pellet the beads I still had some white stuff floating. Anyway, the way I now think I got around this problem is by eluting off of my beads by 2 10 minute incubations with agitation at 4C with 100 mM glycine pH 3 (for example : add 45 uL of 100 mM glycine pH3 --> shake in the cold room for 10 min --> centrifuge to pellet the beads and transfer the supernatant to an eppendorf. Then repeat this and add pool the supernatants). I immediately added 1/10th vol of 1 M Tris pH 8 to neutralize the eluted proteins (in this example I added 10 uL of 1 M Tris-Cl pH 8 to get 100 uL total elution volume). The elution was very efficient (I hardly had any antigen on my beads after elution), and I think I completely got rid of my smearing problem (cross my fingers)!

---