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Large protein will tend to have an increased depletion attraction induced by polymers like polyethylene glycol (see for example Vivares and Bonnete (2002) Acta Cryst D58:472 or Atha and Ingham (1981) J.Biol.Chem. 256:12108)). This effect will be increased with membrane proteins since we are cristallising a protein-detergent complex with a larger radius than soluble protein with comparable masses. One can simply repeat the screen after dilution of the reservoir with water (1:1).
Daniel Robert Cleverley a écrit :
*** For details on how to be removed from this list visit the *** *** CCP4 home page http://www.ccp4.ac.uk *** 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.
