Phenyl Fast Flow Column

[Bryan Macilko]

I am having resolution problems using an approved production protocol. I'm running a Phenyl Fast Flow column to purify calmodulin and the pooled protein needs to be twice as concentrated than I am obtaining in order to be usable for the next step in production ( cross-linking to a resin ). Both myself and another person have had the same concentration issues. Others who have successfully made the product have been able to elute in less than half the fractions than we do.
Technical information:
phenyl sepharose 6 fast flow resin column: 300 ml column run at room temp. on an Akta
equil. buffer: 40mM Tric-HCL,6mM CaCL2,500mM KCL,1mM DTT,buffer adjusted to pH 6.0
elution buffer: 40mM Tris,6mM EDTA,500mM KCL,1mM DTT, buffer adjusted to pH 6.0
Provided that the starting material bio-mass is fine, what conditions would account for the two of us having the same concentration issues?
Thanks.

[tom jupille]

The first thing that comes to mind would be a problem with the column (maybe a void space or an improperly packed column) resulting in loss of plates and resulting broadening of peaks.

Do you have a standard procedure for checking the column efficiency?