Jhdaivs::SumoPrep

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(New page: ulp1 has c-term his tag for purification expressed from pET24d in rosetta - (DE3) pLysS - grown in lb/2xyt/cam/kan - 1 L - induced 1 mM iptg 4 hours resuspended in 20 mM tris pH 8.0, 15...)
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Revision as of 17:36, 3 February 2009

ulp1 has c-term his tag for purification

expressed from pET24d in rosetta - (DE3) pLysS - grown in lb/2xyt/cam/kan - 1 L - induced 1 mM iptg 4 hours

resuspended in 20 mM tris pH 8.0, 150 mM NaCl, 10 mM BME, 10% glycerol, 10 mM imidazole

2x freeze/thaw, lysozyme, sonication NO PMSF!!! spin at 14 k 40 min, apply to 1 mL ni-nta column, wash, elute with lysis+300 mM imidazole. run on gel filtration (s100) column in 75 mM tris pH 8, .5 mM DTT, 1 mM EDTA - concentrated and saved with 50% glycerol at -80 (20 uL aliquots). once thawed used at 4 c and never re-frozen.

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