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
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 | 16 | |
| MW | 54 | FT | 54 | FT | Ruili Cre | 67 | 65 | 63 | 61 | 59 | 57 | 55 | 53 | 51 | 49 | FT |