Many brands of pH paper give 0.5-1 pH reading lower for Hepes buffer. pH meter is more reliable to measure the pH of Hepes buffers. Tris pH increases in cold. If TCEP is used as reducing agent, make sure the stock solution has been pH-adjusted. --Chun
-----Original Message----- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Jacob Keller Sent: Thursday, October 11, 2007 10:03 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] His tag does not bind. Recently, a make-or-break difference for me was making sure the pH was what I thought it was: In the presence of 50mM HEPES pH 7.0 (at room temp), the pH of my protein sample was actually nearer to 6 at 4degC, as judged by pH paper. I am not sure whether the pH difference was mainly because of the temperature drop or because of other components in the mixture, but adding an additional 50mM TRIS pH 8.5 (RT) made all the difference. The pH paper after this addition indicated a pH of 7-8. In the former case, the protein was completely undetectable in elution fractions, and in the latter, was successfully purified. Of course this makes complete biochemical sense, but nevertheless I am very glad I checked the pH. Jacob Keller On Wed, 10 Oct 2007 8:12:46 pm CDT changrui lu wrote: Dear all, I am trying to express a 150 kd protein in E coli. I have it in two constructs, one with pmal-his and other with only his tag at N terminus. The full length protein can be detected both by sds and western using anti-his (190kd and 150kd respectively) but strangely neither binds to his-column very well. The majority of the full length comes through the column either at loading step or low salt wash step. The major species that gets trapped and eluted is the mbp-his truncation (~40kd). Some, though very little, full length protein did make it out the his column. The pmal-his construct does not bind amylose resin any better with majority flows right through. All purification are carried out under standard conditions as mentioned in the manuals. The protein is soluble and does not precipitate in the columns. I appreciate and ideas or explanations. Thanks in advance. Ray Cornell Univerisity ===========End of original message text=========== *********************************** Jacob Keller Northwestern University 6541 N. Francisco #3 Chicago IL 60645 (847)491-2438 [EMAIL PROTECTED] ***********************************
