Dear Meg, Please note that there are many ways to do HIC. Different resins result in different degrees of separation and it's unfortunately impossible to predict what would happen without trying it out. Which is why various companies sell HIC 'trial kits' - a few little columns packed with media substituted with various chains - so that you can try on a small scale. Hopefully you can lay your hands on one of those kits or on something similar.
As others pointed out, HIC elution peaks are not normally sharp - this is expected given the nature of the interaction between the matrix and the protein. The resolution issue is either a) due to incompatible resin b) because even your 'good' protein might be not entirely folded after all or c) because you've not found the right kind of salt/additive combination to do the HIC om. Notably, pH plays a role as well. If as I pointed out earlier, your protein survives transition from low to high pH this might open doors to other purification venues. As far as buffers for pH 5.0 - Citrate or MES should work, IMHO. Artem -----Original Message----- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Meg Sent: Friday, September 26, 2008 4:05 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] HydrophobiC interaction Chromo Dear all, In continuation with my earlier mail on problem in cation exchange chromato, i was suggested to go for hydrobic interaction chromato [HIC] as an additional step. my protein is indeed a hydrophobic protein. we have sepharose 6 fast flow low sub resin and my protein has a pi of 5.8 -6.3. one of the patent papres relating to the same moleculte it was mentioned HIC in suitable buffer containing 0.5 M NH4SO4 pH of 5.0 and elution in same buffer without NH4SO4 pH 5.0. They have mentioned addition of 2-20% ethanol in the elution buffer. is it O.K. to add ethanol in buffer. Can anyone provide me a suitable buffer recipe to use at pH 5.0 for HIC. i performed the HIC but the resolution was not good and my sample was diluted 10 fold and elution was near end of elution gradient. Kindly comment thanks in anticipation
