Paul.xu at 17:40, 2 June 2019

2019-06-02T17:40:25Z

New page

= Purification of T4 Polynucleotide Kinase =

'''Clone '''#: 

'''Host;'''

== 1. Expression ==

== 2. Capture and Purification ==

Ni

== 3. Purification using Capto SP ImpRes ==

*Dilute protein from Ni column with 10 vol of 50 mM NaCl,  20 mM potassium phosphate buffer, pH 7.0
*Load to 20 ml Q-FF (1.6 cm id) coupled with 25 ml Capto SP ImpRes (1.6 cm id) using super-loop
*Wash with 150 ml of 50 mM NaCl,  20 mM potassium phosphate buffer, pH 7.0
*Reverse Q-FF column.
*Linear gradient from 50 mM NaCl to 1.5M NaCl, in 20 mM potassium phosphate buffer, pH 7.0, flow rate 1.5 ml/min, 400 ml gradient, 10 ml/fraction

T4PNK eluted at 15% B, 220m M NaCl

[[File:T4PNK SP.JPG|RTENOTITLE]]

{| border="1" cellspacing="1" cellpadding="1" style="width:500px;"
|-
| Lane
| Desc
| Note
| 
|-
| 1,2
| PNK from Ni column, step gradient
| Lot 160-1764 
| 
|-
| 3,4, 5
| PNK from NI column, step gradient, then GE haparin column
| Lot 160-1764 
| 
|-
| 6,7
| Cre from NI, then QFF-SP FF.
| Lot  161-1010 
| 
|-
| 8,9
| Cre from Ni,  QFF-SP FF,  then GE haparin column
| Lot  161-1010 
| 
|}

[[File:PNK Cre QC 6 1 19.jpg|RTENOTITLE]]
[[Category:Protocols]]

Purification of T4 PNK - Revision history

Paul.xu at 17:40, 2 June 2019