Aggregation Prone Protein Purification


I am trying to purify an aggregation prone hydrophobic protein. I have had lots of problems with having it coat my C18 semi-prep column and it tailing as it elutes. To get all of it off the column and prevent pressure build up (which we are worried is effecting the pumps on our HPLC system and is causing shifting peaks retention time) I need to flow lots of denaturant through the system. I am worried about how much denaturant I am using on my system and wondering if someone could provide suggestions on how I might better purify my protein on RP-HPLC. Would a different mobile or stationary phase help? The method I use is 20 to 80 % Buffer B over 40 mins with a flow rate of 3.7 mL/min with Buffer A= 99.9% Water +0.1%TFA and Buffer B= 99.9% Acetonitrile + 0.1% TFA.

Thanks, Sam

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