Comparative Physiology @ Guelph
Solutions for Phenylsepharose Cl-4b Column-Sigma or Pharmacia
Buffer 1-Equilibration       1 Liter            4 liters
25 mM Tris pH 7.5             25 ml 1 M Tris    12.11g
0.3 M KCl                         22.37 g            89.47g
5 mM CaCl2 Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â 5 ml 1 M CaCl2 Â Â Â Â Â Â 2.94g
0.5 mM DTT Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â 0.5 ml 1M Â Â Â Â Â Â Â Â Â 2ml 1M
Buffer 2-Wash               1 Liter
25 mM Tris pH 7.5 Â Â Â Â Â Â Â Â Â Â Â Â 25 ml 1 M
0.1 M KCl                         7.46 g
5 mM CaCl2 Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â 5 ml 1M
0.5 mM DTT Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â 0.5 ml 1 M-add just before using
Buffer 3-Elute                1 liter
25 mM Tris pH 7.5 Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â 25 ml 1 M
0.1 M KCl                                       7.46 g
20 mM EGTA or EDTA Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â 40 ml 0.5 M
1 mM DTT Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â 0.5 ml 1 M
Column Regeneration Buffer-after sample runs through to wash column
6M urea
You must wash this out very very well after using with Buffer 1
At least 6-7 column volumes of Buffer to wash out urea
Tips from Charles
Dialyze your protein into buffer 1 before loading column
Equilibrate column with 2 volumes buffer 1 before loading protein
Wash (buffer 2) until base line (dropped) returns to starting point level
Elute until you see peak coming up and returning down to starting point
Scan should see peak at 276nm and “knuckles†on downward slope