I'm trying to pulldown a FLAG tagged protein. The protein tagged is well overexpressed in my cell culture (10cm diameter dishes) (detected by wb and silverstain). I extract my protein with 2,5ml of lysis buffer (50mM tris-HCl with 500mM NaCl pH 7.4) per dish, 2H at 4°C (i've also tried over night). But when i perform the IP with flag agarose beads or flag magnetic beads (binding over night 4°C with the protein complex) I have nothing in the eluate. I checked the washing steps by WB and I tried different elution buffer (FLAG peptide competition at 100µg/ml five one volume column; acetic acid with acetonitril; glycine). I detect my positive control (BAP-FLAG fusion protein at 4µg) in the eluate but not the protein of interest.
Does anyone have an idea about wgy it doesn't work?
Hi, can be several problems. First, quality and type of Ab used for IP. I usually use Flag M2 antibody (Sigma) for IP experiments, works fine. Some Ab quality is not high enough to be used with IP. Also, try and use the following sequence of IP steps: Lysis, addition of Ab (no resin or magnetic beads at this point), incubation (for 3-4 h or O/N at +4oC), addition of either A/G resin or magnetic beads followed by 2-3h of incubation at +4oC, elution (either under denaturated or native conditions). Sometimes, when you add Ab coupled to the resin the IP efficacy drops down due to sterical blockage of Ab epitope. Second, in some rare cases the antigen (Flag tag sequence or any other taget sequence) is not exposed under the native conditions (IP) but is exposed when the protein is denaturated (as in the case with WB). If you have any info on the structure of the target protein, make sure that the terminus used for the fusion is solvent-exposed. Finally, I personally do not like the buffer composition for Lysis you described and I assume you use it also for IP. It should contain a solubilizing component, let say, NP-40, from 0.1 to 0.5% final, as well as some other additions (Glycerol, 0.1-1 mM EDTA, KCl sometimes). NP-40 helps in preventing protein denaturation and aggregation.
Good luck.
How much antibody you are using to pull down the protein and the company? Is it specific for IP?
Hi. I'll try with denaturing condition. Thank you for your suggestions.
I use 150 µl of beads precoated with anti FLAG M2 antibody from Sigma. it seems to work for IP as my positive control is detected after the ip.
When you did the western to check the washing steps, did you run the beads themselves? Then you'll know whether your protein isn't binding to the beads, or whether it is binding but failing to elute.
I keep the washing solutions to check if my protein of interest isn't lost during these steps. It's not. I also run the beads themselves but i only detect the heavy and light chains of the antibody. As a lot of my protein is still in the solution after the incubation with the beads I think that binding step doesn't work.
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