have you tried playing with your stock detergent concentration?
By mixing your stock protein with the crystallisation buffer, the dilution of the LDAO may take it below a critical concentration for your protein, hence the precipitation.
Alternatively you could try using the lower protein concentrations such as 2mg/ml but use a different protein:buffer ratio in the drop... 2 protein : 1 buffer, or variants thereof.
HTH,
Dave
On 28/09/06, Robert Cleverley <[EMAIL PROTECTED]> wrote:
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Hi,
I am trying to crystallize a c. 200 kDa pentameric membrane protein of 40 kDa
subunits, purified in LDAO detergent. It is soluble (not pelleted after half
hour spin at 100,00G) at 10 mg/mL. However, using the Molecular dimensions
Memsys/start screen, it immediately precipitates on mixing with the
precipitants in each well, even when the protein is diluted down to 2 mg/mL and
the precipitant diluted two fold. Now I'm hesitating about the best thing to
do - try and do all screens at 2 mg/mL protein concentration or less (which, at
10 uM, seems unusually low from what I've read) or to stick with more typical
protein concentrations for protein concentration (i.e . 50 uM as recommended in
the MemSys/start instructions, which is 10 mg/mL for my protein) and just
dilute the precipitant as much as possible.
Any suggestions welcome!
Rob.
--
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David Briggs, PhD.
Father & Crystallographer
www.dbriggs.talktalk.net
