I have not used, during protein prep, more than 10% glycerol.
However, I did follow one protocol, at the end of the prep, to store the
purified enzyme in 50% glycerol at -80 degree frozen. I guess the percentage
of glycerol is not much a concern as long as your procedure/instrument
can endure the high viscosity/back-pressure. I am wondering though
about your rationale for increasing the glycerol. Are you suspecting
hydrophobic, non-specific stickiness of the protein be the culprit for your
problem? Is there other reasons that might cause the issue you observe?
disulfide bond formation? ionic interaction? protein unfolding? etc.
Practically, there are many steps/details that can be altered from one
prep to another, even though one may not realize it. I had many such
experiences, personally and from colleagues. It sure helps to back track
every little details of your operation to trouble-shoot. Hope you can
get over this hurdle soon. Good luck.
Ligong Wang
Yale University
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