I have incubated my protein with thrombin, and it has successfully cleaved the tag from my protein. I was thinking that the thrombin should have show in the SDS-PAGE as well, but I didn't see any band for it. I am so curious about the reason, pleaseshare your thoughts with me! =D Thank you!
What was the ratio of thrombin to target protein? Was it high enough that you would expect to be able to detect the thrombin band, given the sensitivity of the staining procedure and the total amount of protein in the lane? Thrombin is a 36 kDa protein. Was the molecular weight marker of this mass or smaller on the gel, or did it run off the bottom?
@Adam B Shapiro I add in 1U/uL thrombin into 1mg/ml protein. Is it too diluted for detection on SDS-PAGE? My marker range from 116kDa to 14.4kDa, so should be able to detect the 36kDa thrombin. I didn't over run my SDS-PAGE as well. Thank you.
Bovine thrombin from Sigma is reported to have a specific activity of >1800 U/mg. Let's suppose your thrombin has a specific activity of 2000 U/mg. If you added 1 U/µl (=1000U/ml), then your sample would have been 0.5 mg/ml in thrombin. If your gel lane included 10 µl of protein sample, then it would have had 5 µg of thrombin. This could easily be detected by Coomassie Blue staining. Try doing this sort of calculation on the sample you ran on the gel to see ho much thrombin was in the lane.
Hi Astelia,
when I did thrombin cleavage I might have seen a slight band on Coomassie stained gels which was, however, nowhere as thick as it should have been considering the amount of thrombin loaded. Some proteins do not stain that well with Coomassie and trying another dye might be worthwhile try (if this of importance for you). However, what I did see was a clear band at about 60kDa which was only present after addition of thrombin indicating a contamination in the commercially availabe thrombin I reckon :)
Cheers
Peter
What was the purity of your sample? If the activity was measured based on the total amount of protein and not the thrombin, then you simply may not have enough thrombin to see because the bulk was a "filler". Silver staining will increase your sensitivity about 10x and you could try it.
I would simply buy a much higher quality of thrombin where the amount of protein is over 80% and try it again. You should be able to see 5 ng of protein which should give you a positive result even if 99.9% of the thrombin was a filler.
Why do you want to see thrombin on the gel? Typically, people are interested in the effect of the protease on their samples. You may check it by testing a commercial substrate or simply a know protein which is cleaved by thrombin. With a commercial system, you could estimate the purity of your thrombin sample while using a know in-house standard would simply give you a yes/no answer.
Hi Wieslaw Swietnicki . Thanks for your comment. Looks like I had some of what you call 'filler' in my thrombin :-)
Cheers
Peter
Hi Peter,
I guess there are plenty of cows in Australia and other countries, Poland included. Extracting "thrombin" from a leftover from a slaughterhouse would be a normal way to get much more money than for the meat. I would not be surprised if the "filler' was actually a lyophilized solution from the ground meat.
Best regards,
Wes
When cleaving your Protein with Thrombin what was your bufffer nd pH conditions. It is possible that even after Thrombin cleaved your protein, that it also began to cleave itself. Thrombin is stable at pH 7.0, however at pH 6.5 it is not stable and will begin to break down in to small subunit components. Any further questions, please review literature cited on the MilliporeSigma Website as related to thrombin or if further questions please contact me.
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