[Babcock_Hall]

Hello Everyone,

We would like to remove thrombin from our protein.  However, they are the same size, and so gel filtration is obviously not going to work.  Given how common thrombin digests are (we use it to remove a polyHis tag), what sorts of methods have people found to be successful?  I know that there is a benzamidine-based column, but it is a little pricey, and I would like to avoid the expense if I could.  If we cannot remove it, we could inactivate it with APMSF.

[Yggdrasil]

Does your protein look pure enough after the digestion or do you plan to do additional purification steps afterwards?  If not, I believe you can buy bead-immobilized thrombin (I haven't ever used this, so I'm not sure how its activity compares to regular thrombin).  If you do need additional purification steps, you could try ion exchange chromatography to see if that works to separate your protein from the thrombin (some people like to use ion exchange chromatography on a Mono-Q column to clean up thrombin from contaminating proteases prior to performing the thrombin digest).

[Babcock_Hall]

The protein looks quite pure after the nickel column and gel filtration.  We have not used immobilized thrombin, but we will keep it in mind.  We are looking into ion-exchange.  Fortunately, the calculated pI of the protein of interest is quite low, and I suspect IEX will work.