1. Since you tried 6 different antibodies, I am sure one of them will be compatible for Western. Make sure your protein of interest is expressed in the lysates.

2. Try Protein-A sepharose because some antibodies do not have much preference for Protein-G or vice versa. There is commercially available Proein-A+G mix which solves this issue totally.

3. Try Longer incubations with Lysate+ProteinA/G beads+Abs

4. Antibody isotype could be one issue. Certain antibody isotypes such as IgG2a/b have some issues with certain beads.

5. It is possible your protein of interest may be around 25 kDa or 55 kDa and masked by antibody light and heavy chains respectively (if yes, try pierce secondary antibodies that do not recognize SDS-PAGE denatured IP antibodies).

6. Try conjugating antibody to beads first and wash off unbound antibodies before IP, which will help to pulldown all antibody bound antigens OR try incubating antibody with lysate first and then add beads (this will help to reduce interefernce from beads against antibody binding to antigen).

7. One need some luck in addition..!! :) Good Luck..!!

I think that CHAPS detergent is little stringent for a cytosolic protein. You could try this buffer : 20 mMTris HCl pH 8, 137 mM NaCl, 2 mM EDTA, 0.1% Nonidet P-40 (NP-40). You could increase the % of NP40 if you need.

Hi,

What is your final aim? If it is to identify protein interactors by mass spectrometry the level/success of enrichment (even in the low 2-20%) can be enough for detecting interacting partners. For this one would need a proper negative control of course. If you couple the IP enrichment with an mass spec readout you would be able to better fine tune the experimental steps.

Low affinity could be the problem, either of the two proteins you are trying to co-immunoprecipitate or of the antibody for its ligand. If so, it might help to keep the solutions as concentrated as possible and minimize the number and volume of washes. Changing the salt concentration could also be helpful, either higher or lower, depending on the nature of the interaction (hydrophobic or ionic, respectively). You might even go so far as to try chemical crosslinking to keep the two proteins together. A disulfide-reversible crosslinker could be used to separate them at the end.

Hello: 

I am not sure I follow. Are you optimizing IP or co-IP? It seems that you are enriching for cytosolic proteins, but have you tried whole cell lysate? If you need to enrich, have you done controls to determine if the fractionation worked? You may be better off using concentrated cell lysate as starting material for IPs. If the issue is co-IP, are you looking for a "known" interaction or for new partners. If the interaction is expected you can try decreasing the salt concentration or cross-linking with DSP. Of course, don't forget to add antibody controls for IP (IgG). Hope this helps. 

1. Since you tried 6 different antibodies, I am sure one of them will be compatible for Western. Make sure your protein of interest is expressed in the lysates.

2. Try Protein-A sepharose because some antibodies do not have much preference for Protein-G or vice versa. There is commercially available Proein-A+G mix which solves this issue totally.

3. Try Longer incubations with Lysate+ProteinA/G beads+Abs

4. Antibody isotype could be one issue. Certain antibody isotypes such as IgG2a/b have some issues with certain beads.

5. It is possible your protein of interest may be around 25 kDa or 55 kDa and masked by antibody light and heavy chains respectively (if yes, try pierce secondary antibodies that do not recognize SDS-PAGE denatured IP antibodies).

6. Try conjugating antibody to beads first and wash off unbound antibodies before IP, which will help to pulldown all antibody bound antigens OR try incubating antibody with lysate first and then add beads (this will help to reduce interefernce from beads against antibody binding to antigen).

7. One need some luck in addition..!! :) Good Luck..!!

Most likely reason: the primary antibody is no good. Check it by western blotting. Check with vendors whether it's supposed to work for IP.  Second most likely reason: your antigen is not well solubilized by the co-immunoprecipitation lysis buffer that you use. Check that by fractionation experiments. It might be that your antigen ends up mostly in the pellet. There are protocols whereby you can solubilise the pellet in a buffer containing SDS, you boo, then you re-dilute back the SDS to allow a low concentration (where IP still works). This way, by doing IP from soluble and insoluble fractions (the insoluble that you solubilise with SDS) you can often find antigens that tend to associate with insoluble fractions. When you use a buffer or the other to solubilise, you must first know where your protein ends up, before trying to IP it. If you don't know where it is, or whether it's there at all, it's a hard experiment to succeed.

I met the same situation like Jenna. After reading several comments above, I think you should do as Sandra suggestion. It makes sense and I agreed with this. I will try to apply to my protein also.

Thank you for all the suggestions above! Just to clarify I am looking for difference in binding partners in the nucleus and cytosol so cannot do a whole cell lysis. My intention is to afterwards send off for mass spec to identify new binding partners. I know that there is plenty of my protein present after lysis (input is run on the western along with the IPs: this is what I have worked out the efficiency from) and I have done IgG controls. The approach I have used so far is to incubate the antibody with the lysate first for 1 hour at 4 degrees and then add the beads and incubate over night in a cold room (usually around 6 degrees). This is what was recommended with the beads but perhaps other incubation times/temperatures may be something else to try. Perhaps as suggested above conjugating the antibodies to the beads will be a good thing to try and I will try some lower concentrations of detergent (and another detergent) to see what happens!

I am agreed with the above suggestions. Furthermore, you could incubate your antibody for example. Anti-flag to the protein A/G agraose beads and preclear your whole cell lysate with IgG. Before addition of pre-clear lysate to the beads, wash the unbound Antobody first from the beads and incubate overnight at 4C. At the last stpe before elution. You could try two ways. Just wash the beads with low salt washing buffer and then elute the protein complex with elution buffer and secondly, wash with high salt buffer and then elute. Remeber to run the high salt sample to make sure your protein complex susceptibility towards high salt washing buffer. It could help you. best wishes

First, why you have decided to co-precipitate! If you use one precipitator does not you gt your desired material? Do you get more material for your analysis by coprecipitation? Otherwise,why you have to do that.Think about it.You will have the answer.

I agree with Adam and Doodwin.

Also, you can try 0.1% tryton x100 for lyzing cells. 

By the way, I have been trying different Abs from Santa Cruz for Co-ip, none of them worked well.

Thanks for your answer. Yeah I'm staying away fro santa cruz antibodies as they haven't got the best reputation for working that well. I've mainly been using CST antibodies. I will definitely try a few things mentioned. All of the answers have been very helpful so thanks everyone!

I think you may have enough things to try at this point, but this guide may be helpful in determining whether your antibodies are best for a protein G or a protein A pull-down (http://www.amsbio.com/brochures/Protein-A-G%20-Affinity-for-IgG-subclasses.pdf). Also, our lab has found that the Cell Signaling Protein A agarose beads (#9863) work well for pull-down in a co-IP.