Editing HTNCre Purification Mep

FPLC purification using MEP column 

<br> 

Date: 3/4/2012 

Experiment by: Paul Xu 

<br> 

5 ml Mep column<br> 

Equilibration: 10 ml buffer A<br> 

35 ml injection<br> 

Wash 15 ml buffer A<br> 

Gradient 10 CV (50 ml)<br> 

10 ml buffer B<br> 

10 ml Buffer A<br> 

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

Buffer B: 50 mM NaoAc, pH 5.0<br> 

<br> 

<br> 

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

<br> 

{| cellspacing="1" cellpadding="1" border="1" style="width: 575px; height: 120px;"
|-
| Sample #<br> 
| 1<br> 
| 2<br> 
| 3<br> 
| 4<br> 
| 5<br> 
| 6<br> 
| 7<br> 
| 8<br> 
| 9<br> 
| 10<br> 
| 11<br>
|-
| Sample Name<br> 
| my Cre 1<br> 
| my Cre 2<br> 
| Recycle Cre<br> 
| Recycle Cre<br> 
| My Cre 3<br> 
| my Cre 1 Flow Thru&nbsp; <br> 
| My Cre FT 2<br> 
| Recycle FT<br> 
| FT 3<br> 
| my Cre 3<br> 
| my Cre 3 FT <br>
|-
| description<br> 
| after MEP column purification<br> 
| after MEP column purification<br> 
| after MEP column purification<br> 
| before MEP purification<br> 
| before MEP purification<br> 
| FT from MEP column<br> 
| FT from MEP column<br> 
| FT from MEP column<br> 
| FT from MEP column<br> 
| after MEP column purification<br> 
| FT from MEP column<br>
|-
| Protein OD280 
| 1.47<br> 
| 0.89<br> 
| 1.316 
| <br> 
| <br> 
| <br> 
| <br> 
| <br> 
| <br> 
| <br> 
| <br>
|}

<br> 

<br> 

E coli: Nico21.<br> 

Plasmid: pET28b-Cre<br> 

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

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.<br> 

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

50 mM Phosphate buffer, pH6.5<br> 

50 mM Phosphate buffer, pH6.0<br> 

50 mM Sodium Acetate buffer, pH 5.5<br> 

Elute with 50 mM Sodium Acetate buffer, pH 5.0<br> 

Clean column with 50 mM 50 mM Sodium Acetate buffer, pH 3.6<br> 

Yield: 80 mg 

<br> 

[[Image:Cre purification mep march 2012.jpg]]<br> 

3/14/2012<br> 

[[Image:Cre_2012_3_8.jpg]]<br> 

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

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| MW 
| 54 
| FT 
| 54 
| FT 
| Ruili Cre 
| 67 
| 65 
| 63 
| 61 
| 59 
| 57 
| 55 
| 53 
| 51 
| 49 
| FT
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[[Category:Protein_Purification]]