Jump to content

How to remove thrombin from a digest


Recommended Posts

Good Afternoon,

 

We would like to remove thrombin from a post-nickel column digest. However, our protein and thrombin have similar molecular weights; therefore, I don't hold out much hope for gel filtration. We think that cation exchange may work, inasmuch as our protein has a low pI. We are interested in co-crystallization experiments with a second protein.

 

There is a benzamidine column, but it is a little pricey. There is also biotin-labeled thrombin, but it has the same drawback. There is also APMSF as an alternative to PMSF, and this would at least inactivate the thrombin. Thanks for any suggestions.

 

Link to comment
Share on other sites

If immobilized thrombin or affinity columns are too pricey I guess you are limited to whatever separation method you have at hand (as new columns are not cheap either). Without knowing your target protein it is tricky to say but at least in theory reversed phase or ion exchange prep HPLC should do the trick. Even if inactive I would try to avoid additional contaminants in crystallization experiments as much as possible. Also, I typically check purity of overexpression purificaitions via LCMS as sometimes stuff stick to the column and get co-purified, which can be a problem if the desired proteins is not very highly expressed,

Link to comment
Share on other sites

Thanks. We were planning to do classical (low pressure) cation exchange, owing to a large difference in theoretical pI values between the two proteins, thrombin and the N-terminal and middle domains from FliM, which was well expressed. I imagine if this fails, we will go with a column of benzamidine or possibly biotin-labeled thrombin.

 

We are now routinely checking proteins with mass spectrometry. This is our first attempt at preparing this construct; therefore, we do not yet have HPLC conditions worked out.

Link to comment
Share on other sites

Good idea. A long time ago, I immobilized alkaline phosphatase myself, but I doubt that it would be worth my time to learn how to immobilize thrombin. Better to go with the commercial product. Any thoughts about which is better, removal of thrombin via a benzamidine column versus immobilized thrombin?

Link to comment
Share on other sites

Create an account or sign in to comment

You need to be a member in order to leave a comment

Create an account

Sign up for a new account in our community. It's easy!

Register a new account

Sign in

Already have an account? Sign in here.

Sign In Now
×
×
  • Create New...

Important Information

We have placed cookies on your device to help make this website better. You can adjust your cookie settings, otherwise we'll assume you're okay to continue.