I have done this (or tried to do this) as well and almost ended up bald from ripping out my hair!! My first suggestion would be to try to get down to a 1/25 or 1/50 dilution. If the background starts to get too high then try blocking the Fc region. You can buy commercially available Fc blockers. I also tried conjugating the antibody with HRP which seemed to help some as well. Good Luck!!
Sarah Goebel, BA, HT(ASCP) Histotechnologist Mirna Therapeutics 2150 Woodward Street Suite 100 Austin, Texas 78744 (512)901-0900 ext. 6912 -----Original Message----- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Barbara Verstraeten Sent: Tuesday, January 04, 2011 8:14 AM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] custom-made polyclonal antibody doesn't stain Dear all, I had two antibodies made (in rabbit) for the same peptide. It was the first time I did this : the firm designed the immunization peptide, injected and bleed the rabbits and purified the antibody. I have tested it on paraffin sections: all background, no staining where it should be. I tried different retriever buffers and detection with dab and fluorescence. I have tested it on western blot: a lot of background, dirty lanes. To get a clearer view I performed a co-IP. Still not really great. Can anybody help me on this issue? I just want it to work for staining. Thanks a lot in advance! Barbara Verstraeten, Drs. Evolutionary Developmental Biology Department of Biology Ghent University K.L. Ledeganckstraat 35 B-9000 Ghent Belgium tel: ++32/(0)9 264 52 31 fax: ++32/(0)9 264 53 44 http://www.evodevo.ugent.be _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet