Paul.xu at 21:01, 6 March 2012

2012-03-06T21:01:20Z

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=== Date:2012-3-1 ===

=== column:HiTrap phenylFF(low sub) ===

=== protein:HTNCre ===

 

== Method 1: ==

1.Equlibrate column with buffer 2M (NH4)2SO4 +20mM Tris-HCl 

2.Load sample 0.5ml buffer (2M (NH4)2SO4 +20mM Tris-HCl) +0.5ml sample 

3.Wash colume with 5 ml buffer 2M (NH4)2SO4 +20mM Tris-HCl into 10 tubes 

4.Elute with 8 ml 1*PBS/10%glycerol into 4 tubes 

5.Test protein with Brandford to observe which tube protein is in 

result: 10 wash tubes -no protein 

          4 elute tubes - have protein, especially in the first tube, the fourth tube hardly has protein,protein can  be eluted completely with 4 column volume elute buffer 

 

== Method 2(high salt condition): ==

1.Equlibrate column with buffer 2M (NH4)2SO4 +20mM Tris-HCl

2.Load sample 0.5ml buffer (2M (NH4)2SO4 +20mM Tris-HCl) +0.5ml sample

3.Wash colume with 5 ml buffer 2M (NH4)2SO4 +20mM Tris-HCl into 5 tubes

4.Washcolume with 5 ml buffer 2M NaCl/20mM Tris-HCl /10%glycerol into 5 tubes 

5.Wash colume with 5 ml buffer 1M NaCl/20mM Tris-HCl /10%glycerol into 5 tubes 

4.Elute with 5 ml 1*PBS/10%glycerol into 5 tubes

5.Test protein with Brandford to observe which tube protein is in

result: protein all exist in last 10 wash wells and 5 elute wells, but very little in each well,protein lose too much ,this method can not be used for protein purification 

 

 

[[Category:Protein_Purification]]

HTNCre HIC Purification - Revision history

Paul.xu at 21:01, 6 March 2012