Jhdaivs::SumoPrep

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2x freeze/thaw, lysozyme, sonication NO PMSF!!! spin at 14 k 40 min, apply to 1 mL ni-nta column,
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.
+
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 - 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
ulp = his6-ulp1(403-621), MW = 25473, e280 = 30034/29910

Revision as of 15:20, 8 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 150 mM tris pH 8, 1 mM DTT, 2 mM EDTA - 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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