I have recently performed cleavage of my fusion protein using Thrombin (Image as attached). However, after running the thrombin treated samples with a control (untreated with thrombin), I could see a band shift about 60kDa, but I could not detect my desired protein band. Instead I could only see bands which are NOT corresponding to the size of my protein. Could this be a sign of a secondary proteolysis?
Additional Information:
1)Thrombin cleavage was performed using 3U of thrombin with 300ug of my purified protein at 20C with a time course (2, 4, 8, and 16 hours), just as recommended by the protocol.
2) -The size of my protein with Nus A tag: ~80kDa
-The size of Nus A tag only: 60kDA
-The size of my protein only: ~19kDa
3) No other cleavage sites are present in the vector or within my insert.
4) Extra residues of amino acids from the thrombin cutting site is only 2kDa, which increases the size of my protein to 21kDa.
Hi Saranpal,
you seem to have some low molecular weight bands (same height than the lowest marker band). It is not uncommon to observe unspecific cleavage by thrombin in sequences containing Pro-Arg repeats... We found for several of our fusion proteins that we do not obtain the expected thrombin cleavage product but smaller bands due to the presence of Pro-Arg repeats. So I would think what happens in your case is that your protein of interst gets cleaved by thrombin into smaller fragments. You can check your sequence to try to identify a possible cleavage site. However this will not solve the problem. What about introducing a much more specific TEV-celavage site between tag and your protein?
Hope this helps.
Cheers
Jens
Dear Dr. Jens,
Many thanks for your comment pertaining my query above.
I will try to incorporate the TEV-cleaving site into my vector and see what happens. A part from this, I will also try cleaving the tag using enterokinase (since the site is present within my vector).
Just curious to know, what do the other 2 bands (the one between the 21 and 31 kDa) represent?
Thanks
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