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).
20mM HEPES-KOH pH 8
15 -per glycerol
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.