Paul.xu at 14:29, 15 March 2012

2012-03-15T14:29:04Z

New page

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 

 

 

[[Image:Mep Cre purification 3 5 2012.jpg]] 

 

{| cellspacing="1" cellpadding="1" border="1" style="width: 575px; height: 120px;"
|-
| 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

 

[[Image:Cre purification mep march 2012.jpg]] 

3/14/2012 

[[Image:Cre_2012_3_8.jpg]] 

[[Image:Cre purification 3 15 2012 2.jpg]] 

{| width="200" cellspacing="1" cellpadding="1" border="1"
|-
|
| 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
|-
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|}

 

[[Category:Protein_Purification]]

HTNCre Purification Mep - Revision history

Paul.xu at 14:29, 15 March 2012