Re: [ccp4bb] Fab:antigen complex crystallization!!!
Hi Ivan, Here is another example of a method to crystallize antibody/antigene complexes. It uses a limited proteolysis step to generate crystals of poor quality, which are then used as seeds for an MMS screening... http://www.ncbi.nlm.nih.gov/pubmed/21536542 Good luck, Alex 2011/7/28 xaravich ivan > Hi everyone, > I have been trying to crystallize Fab:antigen complex( 50kda:90kDa) complex > and initially got needle clusters which after microseeding gave me single > crystals but they are very small and I could not repeat the results. I have > been using HEPES buffer at pH 6.8 to do the final SEC purification step of > the complex before setting trays. > I was wondering whether there are some other buffers (that one could > suggest eg tris-hcl etc) which have given decent positive results when > crystallizing Fab complexes.Though I have gone through individual papers > (case by case) to get some idea, It would be great if anyone could direct me > to a comprehensive literature towards studying the crystatllization > conditions of Fab complexes. > Equally, people who have considerable experience could suggest a list of > must do steps for such problems which have routinely been practiced in their > lab > > > Also what is a good storage condition for the excess complex that you want > to use later? > > I would really appreciate any suggestion,help, direction. > > Thanks > ivan >
Re: [ccp4bb] protein lost activity after size exclusion chromatography
Hi Harvey, Could it be that the activity you're measuring comes from a contaminant? Did you test the other fractions from SEC or IEX? Cheers, Alex 2011/3/16 Harvey Rodriguez > Dear all, > > Recently, I came across an obstacle on the purification and acitivty > measurement of my protein. My protein was expressed with an C terminal His > tag in the HEK 293T cells and purified by nickel affinity, anion > exchange and size exclucion chromatography. For every purification step, I > preserved some sample to test the activty. Strikingly, the protein retains > activity after nickel affinity column even for three days but lost almost > all the activty immediately after Mono Q and SEC. Therefore, I speculated > that something (metal ion or co-factor) binding to the protein was striped > by the Mono Q column. Then I skipped this step and only use the SEC for > further purification. However, the protein is still not active no matter > what buffer I use, eg. Tris,Hepes or PBS. The protein I purified by nickel > column is also in the PBS buffer and no additive was added. Buffer exchange > in the concentrator doesn't affect the activity of the protein. Can anyone > explain why anion exchange or size exclucion chromatography destroy the > activity of the protein? Any comment or proposal is appreciated! > > Harvey >
