HTNCre Purification Mep

FPLC purification using MEP column

Date: 3/4/2012

Experiment by: Paul Xu

5 ml Mep column

Equilibration: 10 ml buffer A

35 ml injection

Wash 15 ml buffer A

Gradient 10 CV (50 ml)

10 ml buffer B

10 ml Buffer A

Buffer A: 50mM Tris-HCl, pH 8.0, 150 mM NaCl

Buffer B: 50 mM NaoAc, pH 5.0

Sample #	1	2	3	4	5	6	7	8	9	10	11
Sample Name	my Cre 1	my Cre 2	Recycle Cre	Recycle Cre	My Cre 3	my Cre 1 Flow Thru	My Cre FT 2	Recycle FT	FT 3	my Cre 3	my Cre 3 FT
description	after MEP column purification	after MEP column purification	after MEP column purification	before MEP purification	before MEP purification	FT from MEP column	FT from MEP column	FT from MEP column	FT from MEP column	after MEP column purification	FT from MEP column
Protein OD280	1.47	0.89	1.316

E coli: Nico21.

Plasmid: pET28b-Cre

Induction: 0.4 mM IPTG, 30 oC o/n.

1 st Purification: 10 ml Ni beads, incubate o/n. Elute with 300 mM Imidazole, 600 mM NaCl, 50 mM Tris-HCl, pH 8.0.

2 nd: Load all eluate from 1st purification to 30 ml MEP column, wash with 90 ml each of

50 mM Phosphate buffer, pH6.5

50 mM Phosphate buffer, pH6.0

50 mM Sodium Acetate buffer, pH 5.5

Elute with 50 mM Sodium Acetate buffer, pH 5.0

Clean column with 50 mM 50 mM Sodium Acetate buffer, pH 3.6

Yield: 80 mg

3/14/2012

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