I want to purify a protein complex with co-IP kit (Thermo-Pierce, resin column), but, at the end, I could neither elute enough specific protein nor visualise by WB using antibody against interested protein. Does anyone mind to give me advice about this problem?
Can you attach your blot? Did you see enough of both proteins in the cell lysate? (parallel blot) and write more about your method to get more help.
Most of the antibodies commercially available contains BSA, gelatin or other storage protein stabilizers which are not compatible with co-IP kit. So first step is to check whether antibody to be used does not have above ingredients.
Dear Ananda,
I detected antibody band on blot and there was no band on IgG line (for control). We estimated that there was a problem in the coupling of Ab (which was produced against interested protein). In case of lysate, yes I visualized my protein. I think I should re-perform that experiment. Some authors advice lenghtening the time for cell lysate-column incubation upon overnight.
I would say so too(I did once, it worked for my proteins!), you may try incubating overnight at cold room and wash the following day 3-4 times before elution.
If you sub-clone your protien of interest into a flag vector you can use 'flag affinity slurry' which is "a purified murine IgG1 monoclonal antibody covalently attached to agarose by hydrazide linkage". It would at least save you having to use the IP kit. I have used them regularly and have no trouble.
Dear Agus, after incubation Ab-complex for overnight, do washing with IP lysis/washing buffer and after two washes, measure the amonut of protein in your washing flow through by Nano drop. when the protein is very little in flow through, just stop further washing. then after washing with 1X condition buffer, also measure the concentration of protein complex in flow through. It is quite possible that your protein is flowing with the conditioning buffer. If it so, then just washing with IP lysis budder is enough or you could wash at the lasr with 50 mM NaCl at neutral pH. After overcoming this problem. You first elute the protein complex with 15ul elution buffer and keep it for 1 minute. then elute your remaining complex by adding 40ul elution buffer and incubate at 4C for atleast 5 minutes. and then do centrifugation for 2 minutes. For better concentration, you could reduce the amount of Elution buffer. Best Regards
Dear Agus,
If you are looking for the co-ip product. First do the lysis in following buffer (25 mM Tris-HCl pH 7.4 or pH8.0 + 100 mM NaCl+50 mM NaF+ 2mM EDTA+ Protease inhibitor + 0.5% Tx100). If protein is soluble then Tx100 is very good but in case of Membrane protein you may have to use 2mM DDM instead to Tx100. Lysis should be done at 4 degree and clear the insoluble fraction by centrifugation at 2000g/15 min. Do the co-ip with ab/beads for 1 hr (maximum 90 min). Wash the beads 4x with same lysis buffer and keep the centrifugation speed same.
Elute the protein by adding 50 ul beads (for 1ug antibody per IP) for 95 degree 3 min.
Load 1/4 sample ip elute in SDS page gel.
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