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Comparative Physiology @ Guelph

Solutions for Phenylsepharose Cl-4b Column-Sigma or Pharmacia

Buffer 1-Equilibration Â Â Â      1 Liter Â Â Â              4 liters

25 mM Tris pH 7.5 Â Â Â              25 ml 1 M Tris Â Â Â  12.11g

0.3 M KCl Â Â Â                              22.37 g Â Â Â Â Â          89.47g

5 mM CaCl2 Â Â Â                      5 ml 1 M CaCl2 Â Â Â      2.94g

0.5 mM DTT Â Â Â                          0.5 ml 1M             2ml 1M


Buffer 2-Wash                     1 Liter

25 mM Tris pH 7.5 Â Â                25 ml 1 M

0.1 M KCl Â Â                                7.46 g

5 mM CaCl2 Â Â                            5 ml 1M

0.5 mM DTT Â Â                            0.5 ml 1 M-add just before using


Buffer 3-Elute Â Â                    1 liter

25 mM Tris pH 7.5 Â Â                                    25 ml 1 M

0.1 M KCl Â Â Â                                                  7.46 g

20 mM EGTA or EDTA Â Â                                40 ml 0.5 M

1 mM DTT 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