i suppose this is not from a thermophile or anything but one could try changing the temperature, we need to do this in one case (up in temp to 37-40C or no binding), but its a very thermostable thing.
is the protein functional after e.coli expression? -tommi Quoting Chun Luo <[EMAIL PROTECTED]>: > Try batch binding. Sometimes batch binding for 30 min is more efficient > than > column binding. If your protein binds DNA, treat with DNase or > Benzonase. > Adding detergent may help as has been suggested. --Chun > > > > _____ > > From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of > changrui lu > Sent: Wednesday, October 10, 2007 6:13 PM > To: CCP4BB@JISCMAIL.AC.UK > Subject: [ccp4bb] His tag does not bind. > > > > 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 > > -- Tommi Kajander, Ph.D. Macromolecular X-ray Crystallography Research Program in Structural Biology and Biophysics Institute of Biotechnology P.O. Box 65 (Street address: Viikinkaari 1, 4th floor) University of Helsinki FIN-00014 Helsinki, Finland Tel. +358-9-191 58903 Fax +358-9-191 59940
