I am planning to perform immunoprecipitations using the Pierce anti-HA magnetic beads from Thermo Fisher (Catalog number: 88836) and I was wondering whether anybody has incubated these beads with their protein samples at room temperature for long periods of time?
I have used these beads in a previous lab but we would always perform the incubation at 4 degrees on a rotating wheel for 2-3 hours. However, the protocol on the Thermo website suggests doing this incubation for 30 minutes at room temperature.
I will be using these samples for mass spectrometry analysis and so, I was wondering if 30 minutes at room temperature would be long enough for adequate binding of HA-tagged proteins or would longer incubation times give better results?
Any information would be a big help, thank you!
I think both works, you could run SDS page to see the pull down protein and choose the better one.
It seems that using anti-HA beads would have some kind of background/nonspecific binding.
As Junjie Shao answered, both conditions are fine. It is best not to overdo binding to avoid aggregations and unwanted preciptation. Although for a procedure like IP, your results with largely depend on how well you wash the beads post binding. The concentration of detergent and salt in your lysate and wash buffer would matter more. It is good to vary a few parameters and check the elutions together by western blotting to know what works best.
I would be more concerned with changes in your samples occurring at RT for 30 min than PD efficiency. That being said, 30 min is on the short side, and you probably leave quite a bit of target in solution, but there could still be sufficient material of interest for analysis (staining your gel will tell you that). Failing to see sufficient material in the PD, some optimization (different buffers, longer PD) will definitely improve your signal to noise ratio.
The answer depends on your sample. Is it a total cell extract? What is your protein concentration? Make sure you add enough protease inhibitors. 30 minutes at RT is quite destructive on proteins.
Gloria Volohonsky the samples will be total cell extracts from Candida cells. The protein concentration isn't known because I haven't extracted the samples yet. Normally we use a buffer that contains a variety of protease and phosphatase inhibitors. I was thinking that as well that a RT incubation could potentially be destructive for proteins, which was why I originally performed the incubation at 4 degrees. However, in my current lab, we don't have the space to do this incubation at 4 degrees.
Dean Frawley, another question is whether you are looking to pull down a complex of proteins, or to just isolate an HA-tagged protein? A protein complex would also be sensitive to prolonged incubation at RT. I guess you could try it out and see whether you have a lot of degradation in the sample.
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