Benzamidine Sepharose and Thrombin

[M.T. Ardah]

Hi All,

 

I am using Benzamidine sepharose beads to get rid of thrombin after GST tag cleavage. Afer I added the beads to the protein solution which contains Thrombin I took samples from the beads and the supernatant to check the purity of my protein in SDS and found that most of my protein bound to the beads. Any one have an aidea how to avoid this problem.

 

Regards,

[CharonY]

This should not happen, unless your protein in question has an affinity to pABA. Did you use commercial columns? And if so, followed the manufacturer's instructions?

[M.T. Ardah]

Thax, Actually I am using in tubes not columns!! and what is pABA??

This should not happen, unless your protein in question has an affinity to pABA. Did you use commercial columns? And if so, followed the manufacturer's instructions?

[CharonY]

Sorry for the lab lingo, pABA is short for p-aminobenzamidine. It is more commonly linked to sepharose and my brain must have filled it in. Whether columns or tubes, it is still affinity purification. But it will depend on the precise protocol and material being used.