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Low Thrombin Cleavage Efficiency Problem


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#1 archit

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Posted 15 August 2009 - 10:01 AM

I am trying to cleave a GST tagged protein(about 35 kDa + 26 kDa GST) i have purified the protein but on thrombin cleavage there is very less yeild of my protein with most of it being uncut. I have tried a range of different temperatures, time, buffer conditions but of no use. Can you help? Thanks in advance.
Regards,
Archit

#2 T C

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Posted 17 August 2009 - 03:15 AM

Hey,

Its always a problem cleaving the tag off as sometimes the site is buried. People suggest mild denaturation but its always a risk as the protein may loose its activity. I would suggest changing to a small tag like the histidine tag, presence of which is tolerated for activity assays.

Best,
TC

I am trying to cleave a GST tagged protein(about 35 kDa + 26 kDa GST) i have purified the protein but on thrombin cleavage there is very less yeild of my protein with most of it being uncut. I have tried a range of different temperatures, time, buffer conditions but of no use. Can you help? Thanks in advance.
Regards,
Archit






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