Hi Careina, BN PAGE can be affected by many factors. Considering the complexity and the chance of winning and the amount of information you gain even when you win, I do not recommend fighting it. BN PAGE, like other gel-based methods, requires that your complex is fairly stable - not having a weak affinity and not a high dissociation rate. Also the complex formation should be compatible with the gel running condition: the pH and salts and other things. Coomassie itself may compete for some hydrophobic surfaces or positively charged residues on the proteins - in such case there's little you can do. If you see a band corresponding to the expected complex weight, then congratulations, BN gel might be your tool (with cautions though as you can also get false positives with BN gel). But if not, then my suggestion is to move to other techniques such as gel filtration, analytical ultracentrifugation, BiaCore, ITC and so on. I had a case in which I could confidently show complex formation with gel-filtration and Biacore, but not with BN gel.
Zhijie From: Careina Edgooms Sent: Wednesday, March 27, 2013 5:24 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] off topic, BN PAGE Hi Has anyone found the coomassie in a BN PAGE to be interfering with the oligomeric structure of their protein? If so, how did you deal with this? Thanks Careina
