Dear Sreetama,
First of all, there are no hard-and-fast "rules" for successful
crystallisation, try changing as many different variables as possible
and go with what works.
Having said that, yes, next I would go for a grid optimisation varying
the pH in 0.2 or 0.5 units over as wide a range as the buffer can
reasonable tolerate at the same molarity, and try different
precipitant concentrations on the other axis.
If you have enough protein, try plates at different temperatures as
well, and different protein concentrations (in multidrop wells you can
do this in the same experiment).
A very important variable is the protein preparation itself, prepare
more protein regularly and try to improve on purity and concentration.
Mark
Quoting sreetama das:
Dear all,
I have a 17 KDa protein that gives crystals in a
condition that has 0.1M bis-tris pH 6.5. The crystals are thin
needle clusters and do not diffract. I have tried additives, but
they haven't improved the crystals. I intend to vary the pH of the
condition.
My questions are-
1. should the buffer be kept the same or can it also be changed (as
long as the desired pH is within the range of both the buffers)?
2. in case of a different buffer, should its molarity be the same as
that of the original one in the crystallization condition?
regards,
sreetama
Mark J van Raaij
Laboratorio M-4
Dpto de Estructura de Macromoléculas
Centro Nacional de Biotecnología - CSIC
c/Darwin 3, Campus Cantoblanco
28049 Madrid
tel. 91 585 4616
email: mjvanra...@cnb.csic.es
