Hi all,
Im performing co-IP with Ha-tag protein lysates as prey. My problem is when i incubate M2 agarose + HA tagged protein lysate OR Ha-agarose+ flag-tagged protein lysate, my protein of interest binds nonspesifically to the beads. Below is my experiment design:
1- M2 + flag tagged protein + wash unbound + HA tagged protein lysate +wash --- WB with HA antibody (it works fine)
2- M2 +no flag tagged protein+ HA tagged protein lysate +wash --- WB with HA antibody ( the HA-tagged protein nonspesifically binds to M2)
When i do reciprocal Co-IP, i still get nonspesific signals from flag antibody ( HA -agarose + no HA tagged protein + flag tagged protein +wash--- WB with flag antibody ( The flagged tagged protein nonspesifically binds to HA agarose)
Interestingly, when i use His-tag and express the same protein and make the lysate in the same cell line, the lysate with the his-tagged protein not nonspesifically binds to M2 and HA beads.
How can i avoid this nonspesific interaction with the M2 and HA. What could be the explanation for this problem?? . Is there anyone using M2 affinity gel and preblocking with BSA works fine?
Thank you very much
hi Yasemin , you can read this
file:///C:/Users/Administrator/Downloads/M2-agarose_manual.pdf
If your resin is sticky - you have the following choices:
(1) block resin, e.g. with BSA - you can determine if that works empirically and be sure BSA (or whatever blocking agent) will not interfere with downstream processes.
(2) change the purification conditions - that is, alter the buffer/pH, salt-type/concentration, detergent type/concentration, etc., such that this non-specific interaction no longer occurs - be sure it does not disrupt what you are actually trying to study.
(3) change resins.
All three are valid solutions, but in your case, to minimize the degree of troubleshooting - I suggest you move to magnetic beads - either pre-conjugated with your antibody of interest or with protein A/G and incubating with your antibody of interest (I suggest mild cross linking in this second case)... then carry out the purification.
My observation (shared by quite many researchers) is that the S/N will drastically improve. If you require additional guidance and specific publications to assist with your experimental planning, please message me directly.
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