Table 2.

Buffer compositions and volumes for metal-affinity chromatography.

Buffer	CYP11 Proteins	CYP17A1	CYP21A2
Ni-NTA	50 mM KPi (pH 7.4)	100 mM KPi (pH 7.4)	100 mM KPi (pH 6.8)
Wash	20% (v/v) glycerol	20% (v/v) glycerol	20% (v/v) glycerol
Buffer 1	500 mM sodium acetate	300 mM NaCl	200 mM sodium chloride
	1% (w/v) sodium cholate	0.2% (v/v) Emulgen 913	0.2% (v/v) Emulgen 913
	1% (v/v) Tween 20		*1 mM p-mercaptoethanol
	5 CV	4 CV	4 CV
Ni-NTA	50 mM KPi (pH 7.4)	50 mM KPi (pH 7.4)	100 mM KPi (pH 6.8)
Wash	20% (v/v) glycerol	20% (v/v) glycerol	20% (v/v) glycerol
Buffer 2	1% (w/v) sodium cholate	300 mM sodium chloride	200 mM sodium chloride
	1% (v/v) Tween 20	0.2% (v/v) Emulgen 913	0.2% (v/v) Emulgen 913
	4 mM histidine	100 mM glycine	12 mM histidine
	0.1 mM ATP		*1 mM p-mercaptoethanol
	5 CV	8 CV	5 CV
Ni-NTA	20 mM KPi (pH 7.4)	10 mM KPi (pH 7.2)	10 mM KPi (pH 6.8)
Elution	20% (v/v) glycerol	20% (v/v) glycerol	20% (v/v) glycerol
Buffer	1% (w/v) sodium cholate	300 mM sodium chloride	100 mM sodium chloride
	1% (v/v) Tween 20	0.2% (v/v) Emulgen 913	0.2 % (v/v) Emulgen 913
	80 mM histidine	100 mM glycine	80 mM histidine
		80 mM histidine	2 mM EDTA
			*1 mM p-mercaptoethanol
	5 CV	6 CV	8 CV

^*Inclusion of β-mercaptoethanol is optional, but should be added immediately prior to buffer use.