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Re: [ccp4bb] improve crystal size and quality -- membrane protein |
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- Protein crystallographyMain steps:- Protein purification- Crystallisation Special:- Programs for crystallography- X-ray detectors Basic tutorials:- Chemistry- Protein - Peptide - Amino Acids Xtal community:- CCP4BB |
CCP4bb navigationCCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999Subject: Re: improve crystal size and quality -- membrane protein From: ddas {- at -} LBL {- dot -} GOV Date: 2007-02-28 Hello Balaji, You may try the following: 1) Try different detergents 2) Use a 100 kDa concentrator after protein purification if possible, to eliminate as much of empty detergent micelles as possible. You probably have a lot of these since the CMC value of LDAO is ~0.023% and mol. wt. of LDAO micelles should be ~17 kDa. 3) LDAO conc. down to ~1-2x CMC 4) Additive screens 5) Purification by SEC before crystallization Thanks, Debanu. Bhyravbhatla, Balaji wrote: > Hello All, > We are trying to crystallize a membrane protein but cannot get the > xtals to grow bigger. Presently we have only thin needles (diffracting > to about 8A). Thus far we have tried to change protein concentration, > LDAO concentration, PEG screen as well as temperature. Have tried > macro and micro seeding as well. > > A typical setup looks like: 10% PEG 6000, 0.1M Citrate 5.5, 0.1M > Li2SO4 and 5% MPD with about 12mgs/ml of protein which has about 0.2% LDAO > > Any suggestions or improvements that we can try would be appreciated. > This construct of the protein is not in the membrane but membrane > associated. > > Thanks > > > > Balaji > CCP4bb navigationCCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999 |
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