I need to cleave the trigger factor from my target protein. The current protein is in buffer containing 50mM NaH2PO4H2O, 300mM NaCl, 250mM Imidazole, pH 8. I want to cleave the trigger factor off the target protein using HRV 3C protease. I understand that I have to do a buffer exchange before cleaving. But what buffer do i use to do this exchange? I'm so confused.
Any help will be appreciated.
Thanks.
Dear Alia Mike
it depends the buffer composition that is better for protease activity:
i'm not an expert of the HRV 3C protease, i'm generally use the hand made TEV which is not so compatible with NaCL and imidazole and therefore i have to perfrorm a buffer exchange pbefore the .but from the HRV 3C protease datasheet
file:///C:/Users/Manuele.Martinelli/Downloads/TB420.pdf
it seems that you can try to perform the digestion in the current buffer.
I suggest to yuo to perform a small scale trial, run an SDS-page ans scale up it in case it work well.
good luck
Manuele
According to one supplier (Thermo-Fisher) the optimal reaction conditions are 50 mM Tris-HCl, 150 mM NaCl, pH 8.0.
Takara has a chart showing how the reactivity is affected by buffer components:
https://www.takarabio.com/documents/User%20Manual/7360/7360_e.v1612Da.pdf
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