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Hi Meg, ethanol is often used in a HIC elution buffer as it is good at breaking hydrophobic/hydrophoic interactions. (Detergents or isopropanol can be used to do the same thing). But, as with all things, tolerance to ethanol depends upon your protein. At pH 5, I would guess that Citrate or Acetate buffers are most useful. See http://www.liv.ac.uk/buffers/ for buffer recipes. As for dilution of your sample, optimisation of the gradient you use can reduce the volume your peak elutes in. Regards, David 2008/9/26 Meg <[log in to unmask]>: > 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 > -- ============================ David C. Briggs PhD Father & Crystallographer http://drdavidcbriggs.googlepages.com/home AIM ID: dbassophile ============================