I assume you separate your complex over a size exclusion before setting up trays ? If not try that and see if you get better crystals.
Jürgen - Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry & Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/ On Sep 9, 2010, at 10:59 PM, xaravich ivan wrote: > 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 >
