Paul.xu at 23:10, 27 September 2012

2012-09-27T23:10:58Z

New page

9/26/2012

Host Nico21(DE3)

induction: 0.3 mM IPTG, 6 hours, 37 oC

(expression is not good: 1 mM IPTG, o/n at 37 oC, or 0.3 mM IPTG, 30 oC o/n.

7 L culture.

Ni column, then gel filtration.

 

Gel filtration

[[Image:1346-A_WSN_33_M15_88E_FPLC_2.jpg]] 

 

[[Image:1346-A WSN 33 M15 88E FPLC.jpg]] Buffer 50 mM Tris-HCl, pH8.0, 200 mM NaCl.

column:

Flow rate: 1.5 to 5 ml., 4ml/tube 

Run 10 ul on Bis-Tris gel

[[Image:1346 protein FPLC 9 26 2012.jpg]]

9/27/2012

[[Image:Flu 2012 9 27.jpg]]

Left gel: 

{| cellspacing="1" cellpadding="1" border="1" style="width: 366px; height: 71px;"
|-
| 
| 1 
| 2 
| 3 
| 4 
| 5 
| 6 
| 7 
| 8 
| 9 
| 10 
| 11 
| 12 
| 13 
| 14 
| 15 
| 16 
|-
| Fraction 
| 20 
| 21 
| 22 
| 23 
| 24 
| 19 
| 20 
| 21 
| 22 
| 23 
| 24 
| 25 
| 20 
| 22 
| 22 
| 23 
|-
| OD280 
| 0.3 
| 0.68 
| 0.80 
| 0.50 
| 0.20 
| 0.30 
| 1.61 
| 2.43 
| 1.33 
| 0.72 
| 0.24 
| 0.13 
| 1.35 
| 2.82 
| 1.98 
| 0.31 
|}

 

combine tube 20, 21,22,23-->OD280=1.6, total ~140 ml 

combine other 

Right gel: 

{| width="200" cellspacing="1" cellpadding="1" border="1"
|-
| 
| 1 
| 2 
| 3 
| 4 
| 5 
| 6 
| 7 
| 8 
| 9 
| 10 
| 11 
| 12 
| 13 
| 14 
| 15 
| 16 
|-
| Fraction 
| C5 
| C6 
| C7 
| C8 
| C9 
| C10 
| D10 
| D9 
| D8 
| D7 
| D6 
| D5 
| D4 
| D3 
| D2 
| D1 
|-
| OD280 
| 0.63 
| 0.68
| 0.69 
| 0.72 
| 0.77 
| 0.84 
| 0.97 
| 1.0 
| 1.1 
| 1.2 
| 1.6 
| 2.0 
| 1.8 
| 1.5 
| 1.2 
| 1.0 
|}

 

[[Category:Protein_Purification]]

1346-A WSN 33 M15 88E - Revision history

Paul.xu at 23:10, 27 September 2012