Jhdaivs::SumoPrep

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Current revision (16:04, 8 February 2009) (view source)
 
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ulp1 has c-term his tag for purification
ulp1 has c-term his tag for purification
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expressed from pET24d in rosetta - (DE3) pLysS - grown in lb/2xyt/cam/kan - 1 L -  induced 1 mM iptg 4 hours
+
expressed from pET23? in ? - grown in lb/2xyt/amp - 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
resuspended in 20 mM tris pH 8.0, 150 mM NaCl, 10 mM BME, 10% glycerol, 10 mM imidazole

Current revision

ulp1 has c-term his tag for purification

expressed from pET23? in ? - grown in lb/2xyt/amp - 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 150 mM tris pH 8, 1 mM DTT, 2 mM EDTA, 75 mM NaCl, 75 mM KCl - concentrated and saved with 50% glycerol at -80 (20 uL aliquots) (final is 75 mM tris pH 8 0.5 mM DTT 1 mM EDTA. once thawed used at 4 c and never re-frozen.


ulp = his6-ulp1(403-621), MW = 25473, e280 = 30034/29910

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