immunoprecipitation recently. My main problem is i am getting very clean and my target protein only (as you see in the lane 3,4,5,6) but when i reduced the NP40 concentration to 0.05 percentage i could see enriched pattern in the IP pull down (lane 1 and 2). but the main problem is that i can same pattern in the control also. I am using my lysis buffer for the washing also (2 times washing with lysis buffer 10 mins at cold room followed by 2 times with 1X pbs). I am Using Mag Strep "type3" XT Beads (Incubation 4 hours in cold room with beads).

  1. Lysis buffer:

    20mM HEPES-KOH pH 8

    15 -per glycerol

    150mM KCL

    0.2 per NP40 (I reduced to 0.05 per)

    1 mM DTT

    1X protease inhibitor cocktail

Can anyone help me with this. Should i change the washing buffer what about the stringency. What are the other factors that could increase the efficiency of the co-IPs. I am trying to optimize the protocol. This is human cells.

enter image description here

  • $\begingroup$ What are you doing your Western for - your IP target protein or a known binding partner? Please give identifiers for your different proteins (i.e., Protein X, Protein Y, etc.) and indicate what exactly the samples, IP antibodies, and WB antibodies are in each lane. Or is this an image of a stained gel? Please label molecular weights as well, it's possible you're just staining heavy and light chain. $\endgroup$ – MattDMo Mar 27 at 18:47
  • $\begingroup$ The gel is silver stained. I know my protein (70kDa) but i am looking for the interaction partners (which i am interested). It is a streptavidin pull down. I have given only the experiments only. From the left to right first two lanes reduced NP40. 3rd to 6th lane reduced 0.2% NP40. $\endgroup$ – Rengaraj Mar 29 at 15:31

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