Hi Ivan, Did you try using a buffer other than phosphate? Also maybe a different pH can help keeping the IgG in solution. Although papain prefers pH 6-7, it is a fairly robust enzyme and will cleave with >20% efficiency in the range of pH4-9 (Hoover S & Kokes E ,1946, http://www.jbc.org/content/167/1/199.full.pdf, and Lowe G & Yuthavong Y 1971, http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1177120/pdf/biochemj00648-0125.pdf). Also the un-beaded form of papain is super cheap. So there is no reason to constrain your cleavage in a condition that your IgG doesn't like. Why not cleave the IgG in the original purification buffer supplemented with EDTA and cysteine?
Zhijie From: xaravich ivan Sent: Thursday, September 09, 2010 10:59 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Fab purification and crystallization Hi CCP4bb, I have two questions regarding Fab purification and Fab-antigen complex crystallization and would really appreciate any input from the experienced board. 1) I have got some hits for Fab-antigen complex (150 kD) but they are all needle clusters. Whatever fine screen I formulate, it always gives me these needle clusters. Are there some better common ways to change needles to single crystals? 2) I have certain IgGs from which I purify the Fab by papain digestion (resin from ThermoSci). One of the first steps is to dialyze the IgG with the digestion buffer,( 20mM Na-phosphate and 10mM EDTA pH 7.0) Over night. I always get 30-60% of the IgG precipitated during this overnight dialysis. I tried to increase the salt by adding 200mM NaCl but of no effect. Have anyone experienced such problem? Is there any thing that could be tried to stop this precipitation. thanks in advance. ivan
