Low Thrombin Cleavage Efficiency

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Matthew R Hildebrandt

I also have this problem but it comes and goes seeming at random. The things I have been checking are:

Age of the thrombin and its storage conditions (single use aliquots at -80).

At least 100-150 mM NaCl in the buffer to ensure cleavage.

How are you purifying the protein--by batch with sepharose beads? or by column

Are you cleaving the protein from the beads or eluting the protein then cleaving then removing the GST by beads or column pass again?

Tanja Waldmann

I gave up with this as I tested thousands of conditions like Matthew said and it never ever worked. Sorry, not really good help.