I have successfully over expressed my GST tagged protein in BL21 by using PQE30 vector and it is binding to Glutathione sepharose beads. In coomassie staining and silver staining, I have got satisfactory results in both but somehow after adding thrombin (Amersham), it is not getting cleaved off from beads. Earlier in our lab people have done it successfully but now its not working.
Problem in thrombin digestion in GST tagged protein purification?
You need to trouble-shoot
1. Is your thrombin still active?
2. is your thrombin site available for cleavage?
I usually elute the purified protein (with glutathione), dialyze, cleave, then repass over the GST column. This gives the thrombin better access to the fusion, and you can monitor cleavage.
cat teeth
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