Hi eveyone,
I m facing a problem with a co-ip.
When I add 5% glycerol in the lysis buffer the A/G protein beads do not bind the antibody efficientrly in solution.
When glycerol is not included the beads bind the antibody after three hours of incubation and it s visible on the membrane after the panceou staining.
Did anybody have the same experience before?
tnx
Hi Patrizio,
Try to troubleshoot your problem using the following guides:
http://www.abcam.com/protocols/immunoprecipitation-troubleshooting-tips
http://www.proteinguru.com/protocols/IP%20guide2.pdf
Good luck
I suggest to minimize glycerol to 1% Glycerol to enhance solubility, maintain hydrophobic interactions and prevent water crystal formation in crystallography. Also, this is happen also when you have high imidazole Conc. in binding buffer, and your protein can not bind efficiently. So,use 5-10 imidazole Conc. to increase binding capacity. but for imedazole condition it is experimental based depends on your protein.
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