Refer to Protein G immunoprecipitation kit (Sigma) protocol. I am sure it will be useful and solve your issue

I wonder if you would get more answers if you post this question to some Molecular Biology techniques topic. The fact that you have "immuno" in the name of the technique doesn't make it "immunology protocol".

I wish I could help you but my experience with IP is quite outdated. I am sure molbio folks would be of more help!

there are multiple IP kits available that come with either magnetic beads or strepavidin beads.  Most times it is easier and better to use Protein A/G beads if you are not sure whether or not your protein is Protein A or G.  I use magnetic Protein A/G beads from invitrogen because you can perform the tissue homogenization in either detergent or detergent free lysis buffer.  You can also perform IP experiments using various lectins or biotinylated proteins.

Generally, I homogenize my tissue and determine total protein using a BCA assay.  I add 1 mg/ml of tissue in PBS and then add the corresponding antibody concentration suggested on the antibody data sheet.  I incubate on a 360 degree rotor for 2 hrs at room temperature.  I then add the corresponding amount of magnetic beads that will bind the amount of antibody that I added previously.  I always add the beads in a little excess just in case.  I incubate the samples with the beads for 30 min at room temperature on the 360 degree rotor.  I place the samples in a magnetic rack, wait 30 sec, and then remove supernatant.  I wash the samples 3 times in 1 ml PBS.  After the last wash, I remove the supernatant and add 80 ul of 2X sample buffer and heat for 10 minutes at 70 degrees.  I then put the samples in the magnetic rack, wait for 30 sec, and then keep the final supernatant.  I snap freeze the samples in liquid nitrogen and store at -80 until running in a gel.  When I am ready to run the samples in the gel, I defrost them and then load directly into the gel.

last thing, what is the sample buffer's ingredients? Thank you, thank you very much for your time all of you :)

I use the 2x sample buffer mixture from biorad cat number 161-0737 and corresponding 2 mercaptoethanol (BME) cat number 161-0710.  If you are making 1 mL of 2X buffer, you add 950 ul of the sample buffer + 50 ul of the BME.  Then you add this mixture to your sample mixture in a 1:1 ratio.  So if you have 40 ul sample, you add 40 ul of this sample buffer mixture.  If you need to load more protein in your gels, you can purchase 4X and 5X sample buffers as well.  Good luck

feel free to get in touch with questions around Dynabeads products (working in R&D for the Dynabeads products in Oslo, Norway ([email protected])