Hello All,
I am attempting to cleave a purified fusion protein with thrombin. I have assembled the following reaction: 11.5ul Thrombin (550 US Units/mL), 13.2ul fusion protein (19mg/mL), 100ul thrombin cleavage buffer (10X), 875ul H2O. Thrombin cleavage buffer composition (1x): 50mM Tris pH 8.0, 150mM NaCl, 2.5mM CaCl2, 0.1% BMe.
I cast the gel with 4% stacking and 6% resolving.
Lane 1, 9, 10 are broad range MW Marker
Lane 2 is an old protein prep (did not visualize)
Lane 3 is uncut (MW 226 Kda)
Lane 4 is 19 hrs cleavage at 4C
Lane 5 is 25 hrs cleavage at 4C
Lane 6 is 44 hrs cleavage at 4C
Lane 7 is 48 hrs cleavage at 4C
Lane 8 is 93 hrs cleavage at 4C
MW Marker Bands: (209 Kda, 124 Kda, 80 Kda, 49.1 Kda, 34.8 Kda, etc)
Desired Cleavage Products:
6xHis-MBP - 43.3 Kda
YFP-Cpf1 - 156.4 Kda
Additional Information:
Thrombin MW - 36 Kda
Protein loading is normalized - 2.25ug total protein loaded all lanes
My issue is that I see multiple bands running between 209 and 124 Kda after cleavage. The bands seem to form in equivalent amounts. There is only one thrombin cleavage site in the construct, however, I do know that thrombin is prone to off-target cleavage. I am curious if there is any way to reduce off target activity of thrombin. I believe that only the larger MW band is my desired protein (YFP-Cpf1) and that the smaller band above 124 Kda is an undesired cleavage product. Any help is greatly appreciated.
Thank you
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