Cleaning Up an Immunoprecipitation - (Jan/11/2008 )

I have been running immunoprecipitations using a polyclonal antibody. I am trying to see if this antibody can be used for IP's ..b/c it has never been tested.

Right now I have been incubating cell lysate (made with RIPA, sitting on ice for 3 hours, vortexing 2-3 times) with primary antbody, then add protein G (for 4 hours) spin down remove supernatent, wash 3x with PBS+, take pellet add 20 ul LLB wlo BME (reducing conditions interferes with protein band on gel)vortex, spin, load soup into gel.

I was thinking that instead of washing with PBS+ I could wash with something more aggressive. I thought I might try Tris pH 8 with 1M NaCl. and also try Tris pH8 with 6M Urea. I would compare all three washes with different buffers and determine which one cleaned up the IP the best.

Does anyone have any suggestions on cleaning IP and if my washing buffers sound effective.

Thanks

You may want to start with preclearing your lysates. You incubate your lysates with rabbit IgG (bound to beads) to clear away any binding that nonspecifically occurs with the species antibody. I don't recommend the use of RIPA though. I think it's just too aggressive of a lysis buffer. I use a simple NP40 (1%) buffer to lyse cells for IP. I also prefer to bind the antibody to the bead, wash well and block in 5% BSA before adding the lysate (rather than adding the antibody to the lysate followed by beads). This helps get rid of nonspecific binding to the bead itself. Your washing is definately an issue as well. PBS just isn't going to do the job. Minimally you wash in the buffer you lysed the cells in. You can make more aggressive washes but 1M salt is WAY to high (Urea is very aggressive and will destroy everything). You will disrupt all the interactions. I start by taking the lysis buffer to 250mM and will go up to about 500mM. Additionally you can add a little more detergent but I suggest you do this in small steps to determine the best conditions. It's frustrating to run the gel and see that you washed everything away! The other idea is to use a wash buffer without the salt. Nonspecific interactions can often be through salt bridges. One quick wash in a no salt (or very low) will get rid of this background as well. You can try the washes alone or in combination as well. I had one IP that required a high detergent (high salt did nothing) followed by a no salt wash in order to remove nonspecific bands. You are just going to have to play around with the conditions and see what works best for this situation. I can recommend a book: Using Antibodies by Harlow and Lane. There are many good hint and tips for troubleshooting and this book covers practically all assays involving antibodies.