Hi,
I’m having some IP issues and I’m (seriously) hoping somebody can help. Basically, I’m successfully pulling down my protein BUT it’s also present in my IgG IP. I’m guessing my washing conditions aren’t stringent enough but I’m slightly worried that this non-specific binding is occurring in the first place. Is this normal? My protocol is pretty simple, it's as follows.. 1. Prepare a heart protein lysate using Cell Signaling lysis buffer (0.1% triton plus usual buffers). 2. Incubate 300 micrograms of protein lysate with Dynabeads (50 microliters as recommended by Invitrogen) already bound to an antibody (2 micrograms total) against my protein of interest overnight. 3. Wash beads three times with PBS (+0.02% tween). 4. Re-suspend beads in 50 microliters laemli loading buffer and run gel/western Thanks!! _______________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods
