HTNCre HIC Purification

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