I don't quite understand your question completely. You have cells attached to primary, attached to secondary all placed into sepharose beads? The problem is the sepharose will bind all three, cells, primary and secondary? I am assuming you want to separate your bound Abs from its analyte (ie separate cells, primary Abs, and secondary Abs from one another) there are a few options. Again, I do not know you end goals. But the following solutions work well to un-attach Antibodies from analytes 1) 50%-70% ethanol, 100mM Glycine pH=2.0-2.7,Citric Acid pH=2.0. Just put your purified cells/primary Abs/secondary Abs into one of the solutions and incubate for 5-10 minutes. The Ab will separate and then based on your goals, you can put the solution on your sepharose beads, or remove the Abs with a Protein A/G sepharose first, which should purify out your analyte fairly well, or run a MW separation filter step (try amicon MW filters). FYI, In the case of sepharose binding, you will likely have to raise  the pH back up.  Another option is pepsin/Trysin/enzyme digestion to remove the antibodies. FYI, the enzyme digest will only work if you are trying to recover whole intact cells and you may lose your surface analytes.     

Hi Miriam,

If I understood your question correctly, you have sorted your cells using antibody staining. Now you want to use these sorted cells in a IP experiment but the sorting antibodies are interfering with your IP as they bind your protein A/G beads.

I think if you used a surface antibody to sort your cells you can wash your cells in an acidic buffer to remove these antibodies. Alternatively, you can add a few extra steps in your IP protocol where you wash your lysate with sepharose unconjucated beads for a few times before adding your actual  IP antibody and beads. In this way your unwanted antibodies will have bound the washing beads which you can easily discard and save your clean supernatant (lysate).

Good luck!

Hi,

thank you for your answers.

I use an intracellular antibody. But I want to lysate my cells after sorting getting the intracellular proteins. The lysate buffer consists of 0.1 % SDS, 1 % Nonident-P40 (Igepal), 0.5 % Na-Deoxycholat and PBS.

I want to do a Co-IP with this lysate to check whether my intracellular protein binds to another protein (=binding protein). I have an antibody against this binding protein. So of this experiments I want no other antibodys in my lysate. Or is it possible to block the FCII region of the FITC-antibody and so these antibody can not bind to the protein A/G beads?

@ Rajaei: If i you use these sepharose unconjucated beads, will my protein (which is binding to the primary antibody) also be washed away or will it stay in the supernatant?

Hi Miriam,

I actually assumed that you have sorted your cells by targeting a different protein than your protein of interest or any possible interactive partners. In this case, I am afraid that you will risk washing away your protein/s of interest.

To be honest, I can't think of anyway to target your secondary antibody only but lets wait for someone else to reply.

All the best with your experiment!

That depends on the affinity of your antibody, but you may try this:

- Grow sorted cells for at least 1-2 days (if you can) so they internalize and eat the antibodies. 

- or series of 5-10 washes in media. Most probably won't work but worth a try.

- or, as suggested before, try washes in low pH glycine buffer (pH 3 or so). May kill your cells or cause lysis and denaturation. 

- it is possible that during the lysis for IP your antibodies will fall off. Or not.

But most probably all of this will not remove the antibodies completely.

As of now, only the membrane fraction of your protein is bound to the antibodies (I assume you sorted non-fixed surface stained cells?). You can preclear your lysate prior to IP with A/G beads to remove the existing immune complexes. You will lose the membrane protein (assuming it was solubilized), but there should be plenty left inside the cell as this is overexpression.

To avoid this situation in the future if possible use the vector that coexpresses fluorescent protein and sort cells based on the endogenous fluorescence.

Still a little confused by what your trying to do. As I understand you permeabilized your cells to label an intracellular "protein X" with an antibody (AbX) so you could sort.  After sorting you lysed the cells and now want to bind "protein X" to a "protein Y". Protein Y is then bound to an antibody specific to "Y" (AbY). So you are making a 4 component complex of AbX bound to protein X bound to protein Y bound to AbY? You then want to purify using out only the AbY using protein A/G? IS this correct? 

I must admit this seems a little complex. Might I suggest a few options. 1) use antibodies from different hosts and use an affinity column specific against your AbY host to separate. 2) disassociate your AbX-Protein X complex with the before mentioned solutions then separate AbX from protein X by size exclusion (see Amicon MW filtration units) before adding protein Y 3) Make an affinity column from your AbY, then you can pass your AbX-protein x-protein Y directly over it.  4) use a size separation column, or an Amicon MW filtration unit, to separate out your complexes after a Protein A/G column purification. The fourth option will likely be the easiest and very effective if you have a size separation column. Option 3 is probably the quickest and work the best, but might be expensive. 

If you directly conjugate your IP antibody to beads, avoiding protein A/G the sorting antibodies will not interfere. I have done that with magnetic tosylactivated beads.  

You simply could pre-clear your lysate with protein A/G beads, as Oleksander suggested. Alternatively, use a biotinylated antibody and streptavidin coated beads for IP. Third, if the antibodies from sorting don't interfere with your downstream processing, just don't care and use a little more protein A/G beads to be sure that all antibodies get captured.

Gertrudis is right. You just need to make sure your IP antibody has occupied all binding sites on the beads ie. all protein G/A is bound to an antibody. The  manufacturer's instruction will tell you the binding capacity of the beads which will help you to calculate how much antibody you need to add in order to have full occupancy.