First you should dialyse your protein into a buffer that is suitable for thrombin, as the Ni-NTA elution buffer is not be the best choice (high salt, reducing agents and imidazole reduce the activity of thrombin). Your buffer should have a pH around 8.4, 150 mM NaCl and 1.5 mM CaCl2. You can either let the cleavage reaction proceed over night at 4oC or at room temperature for 2 to 8 hours. My proteins used to precipitate significantly during thrombin cleavage if I kept the sample on a rotator. Leaving the sample alone and simply inverting the container every now and then seemed to work much better. It's difficult to tell when the thrombin has cleaved enough of your protein, so it's best to run a time course and take a sample from the cleavage reaction every 30 or 60 minutes and run it on a gel when you use thrombin on a new protein for the first time.

After cleavage run your sample through Ni-NTA again. This time the cleaved-off His-tags will stick to the beads/column and your protein will be in the flow-through instead of bound to the column. Also, any contaminants that stuck to the column together with your protein during the previous Ni-NTA step will stick to the column again, giving you a purer protein sample in your flow-through. If your cleavage wasn't completely successful, the protein molecules that weren't cleaved will also stick to the beads/column leaving only cleaved molecules in the flow-through. If your thrombin isn't His-tagged, it will also be in the flow-through, but you can remove it either by ion exchange chromatography or size exclusion chromatography. As Dmitri mentioned, you can also use beads that have thrombin immobilised on them, so you don't need extra steps to remove the thrombin.

Now to the downside of thrombin: if you use an excess of thrombin or let the cleavage reaction proceed for too long, it will not just cleave off your tags but also randomly chew up your protein. This is the reason why many people have stopped using thrombin (I'm among them) and have opted for other proteases with more stringent and specific cleavage sites as TEV, HRV-3C, PreScission or Ulp1 (SUMO protease). Also, you can generally produce these other proteases yourself, which is MUCH cheaper than buying them, while you can't make your own recombinant thrombin.

Hi Esha, you can kill thrombin by adding APMSF (4-Amidinophenylmethanesulfonyl Fluoride) or TLCK (tosyl lysine chloromethyl ketone).

Look for Thrombin Sepharose Beads. Otherwise, gel filtration or other types of chromatography can be a method of choice.

First you should dialyse your protein into a buffer that is suitable for thrombin, as the Ni-NTA elution buffer is not be the best choice (high salt, reducing agents and imidazole reduce the activity of thrombin). Your buffer should have a pH around 8.4, 150 mM NaCl and 1.5 mM CaCl2. You can either let the cleavage reaction proceed over night at 4oC or at room temperature for 2 to 8 hours. My proteins used to precipitate significantly during thrombin cleavage if I kept the sample on a rotator. Leaving the sample alone and simply inverting the container every now and then seemed to work much better. It's difficult to tell when the thrombin has cleaved enough of your protein, so it's best to run a time course and take a sample from the cleavage reaction every 30 or 60 minutes and run it on a gel when you use thrombin on a new protein for the first time.

After cleavage run your sample through Ni-NTA again. This time the cleaved-off His-tags will stick to the beads/column and your protein will be in the flow-through instead of bound to the column. Also, any contaminants that stuck to the column together with your protein during the previous Ni-NTA step will stick to the column again, giving you a purer protein sample in your flow-through. If your cleavage wasn't completely successful, the protein molecules that weren't cleaved will also stick to the beads/column leaving only cleaved molecules in the flow-through. If your thrombin isn't His-tagged, it will also be in the flow-through, but you can remove it either by ion exchange chromatography or size exclusion chromatography. As Dmitri mentioned, you can also use beads that have thrombin immobilised on them, so you don't need extra steps to remove the thrombin.

Now to the downside of thrombin: if you use an excess of thrombin or let the cleavage reaction proceed for too long, it will not just cleave off your tags but also randomly chew up your protein. This is the reason why many people have stopped using thrombin (I'm among them) and have opted for other proteases with more stringent and specific cleavage sites as TEV, HRV-3C, PreScission or Ulp1 (SUMO protease). Also, you can generally produce these other proteases yourself, which is MUCH cheaper than buying them, while you can't make your own recombinant thrombin.

hi all an thank u for your responses. my protein is in a vector which has a thrombin cleavage site, hence for this one i need to use thrombin. i actually need to cleave off the tag and then check the activity of my protein to ensure if the tag and its associating protein was interfering with the activity.

can anyone suggest if ho much of thrombin to protein would be best for room temperature and how long to do it without typically having to test on a gel after every 60 mins.

Hi Esha, I think Dmitri has the best solution for separating thrombin from your protein solution, namely by using immobilized thrombin:

http://www.biovision.com/thrombin-sepharose-beads-9324.html

As far as how long you have to incubate thrombin wit your fusion protein for optimal cleavage, I don't know. 

Every fusion protein is different in terms of how well it is cleaved. 

Hi Esha,

We run a time course as standard whenever we used thrombin on a new protein because you never know how long it's going to take for most of the protein to be cleaved. After you've done this once for a given protein, you obviously won't need to do it again. How much thrombin you need to add to your protein will vary from manufacturer to manufacturer and from protein to protein (just like the time needed to cleave most of it). A very rough estimate is 1 U per 1 mg of protein (use a bit more thrombin if your protein is particularly small as you'll have more protein molecules in 1 mg and less thrombin if your protein is very large as you'll have fewer protein molecules in 1 mg). 

Hi there,

To remove soluble thrombin you can also try benzamidine sepharose. And I do agree with Antonio : each protein to cleave is a particular case for which you have to define the best condition for thrombin cleavage... 1U per mg overnight at room temperature is generally a good start.

thank u 

If you use biotinylated thrombin, you can remove it using Streptavidin agarose.

I wondering which is the isoelectric point of thrombin?? because I have a problem with the ion exchange chromatography . Thrombien and protein continue toghether after the cromatography

I did 22 degrees for 16 hours can cleave ~90% of my protein.