MBIO 2420 Lecture Notes - Lecture 10: Chromatin Immunoprecipitation, Sepharose, Immunoprecipitation
7 Dec 2016
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Don"t use an antibody - use a tagged protein to gst. Use glutathione agarose beads that binds to gst. Use to purify proteins that bind to this protein: co-ip = co-immunoprecipitation. Pulls down proteins that are bound to pol. Detect any proteins that bind to your protein of interest: chip - chromatin immunoprecipitation. Isolate any chromatin that is associated with your protein of interest. Crosslink protein - dna complexes with formaldehyde. Or treat with enzyme to break up chromatin. Scan for known targets via pcr amplification of chip dna fragments. Problem: antibodies are showing up in detection because protein is bound to much. Cross link antibodies to beads - protein a/g sepharose - so antibodies cannot leave beads so it"ll let go the proteins. Prior to addition of antibody - step 3. Advantages: decreased unbound ab in lysate = higher antigen recovery, can now use same antibody (same species) for ip and western detection.
