I am very happy to get so many suggestions to purify my protein.
I didnĄt run again HIC because after three times I thought better to try
something else. (but I will do it later)
I have tested the dye reagents (not in a column but in a tube) and the
results were not amazing. I also tryed the hydrylapatite. For the test I
put aproy. 100µl HA in a tube with protein extract and P-buffer 100mM, i
centrifuged and then I eluted with P-buffer 100mM + NaCL 1M, I got some
activity in both steps so I guess that the bounds to the HA, but I
utilised a less amount of HA. The idea was to start a run with 0% salt
and increase untill 1M. I have not run the column yet but, Gautam, do
you think it is better to increase the concentration of P instead of
NaCl? Remenber that the protein has a pH optimum 9, I read that for such
proteins it must be one in the other way round.
I will let you know how it works.
Angela
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