Hi Abdul Yes, I had the same bad experience with some of the Fabs I was preparing by papain digestion several years ago. By the time I just walked around it by starting with more antibody, yields were less than 20% of the starting antibody mass (bad as compared to the 60% from the good ones).
I didn't try this myself, but I believe you can test different buffers for the digestion in small scale (varying salt concentration and pH - rather going up - a little bit). The loss in papain activity can be partially compensated by adding more enzyme. I wouldn't change the antibody nor the L-Cys concentrations. Carlos Em 24/12/2012, às 03:39, Abdul Khan escreveu: > Hi everyone. > We are trying to make Fabs from monoclonal IgGs, we are using insoluble > papain for digestion, to our surprise the protocol we were using gave > precipitates with some antibodies but not all, we have tried 4, Two were fine > and the other 2 just precipitated during the digestion. Our digestion > reaction contains following, > 50 mM Na-Acetate pH5.5, 1mM EDTA, 50mM L-cysteine, no salt, antibody > concentration 1-2 mg/ml. > We appreciate if someone has such experience and would like to share some > expert views, > thanks a lot, > abdul > > -- > Abdul Ghafoor Khan > University of Vienna, > Inst. Med. Biochem., Vienna Biocenter (VBC), > Dr. Bohr Gasse 9/3, > A-1030 Vienna, Austria, > Tel: 0043 1 4277 61631,(lab) > Tel: 0043 69910695081, (mobile) > Email:abdul.ghaf...@univie.ac.at
