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文献和实验.27. Column Buffers Make 500mls equivalent of base buffer: 10ml 1M Hepes pH 7.9 100ml 10% glycerol 5ml 10% NP40 (or 5.0ml 300mM Octyl glucoside) 200ml 0.5 EDTA ------- 115.2ml /5 = 23ml Base 1M KCl2.5M KCl dH20 for 0 KCL 46ml 0 0 154ml = 200
A280.Once absorbance has leveled off at zero,the column is sufficiently washed. 10)Elute column with the appropriate salt solutions. - When eluting SMase activity,elute: a)three times with (1 ml of)100 mM NaCl. b)two times with (0.7 ml)of 250 mM
in the column matrix!! 8)Wash column with 5 column volumes of lysis buffer containing no DMSO!! --> NOTE: DMSO strips proteins off of the coulumn! 9)Run a 40 ml salt gradient collecting 1 to 2 ml fractions: - Start at 0 mM NaCl and end with 500 mM NaCl. 10
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