Thanks Nikolay
I am doing on column cleavage, so I am incubating it
overnight at 4-degree C. Do you think even on column digestion is also going to
be done in one hour.
Thank you
Dhiraj
________________________________
From: CCP4 bulletin board <[email protected]> on behalf of Nikolay Dobrev
<[email protected]>
Sent: Thursday, February 3, 2022 11:53 AM
To: [email protected] <[email protected]>
Subject: [External] Re: [ccp4bb] protease inhibitor and 3c protease
Dear Dhiraj,
the 3C protease is from the Cysteine protease family.
Therefore for sure, you can add EDTA (if it is not indented for follow up IMAC
purification) and that will inhibit most of the metalloprotease. Furthermore,
you can resort to Serine type protease inhibitors like PMSF or Aprotinin.
One side not, the 3C cleavage does not need to be long, as it is an extremely
potent protease. If the cleavage sequence is well accessible in less than 1
hour at 4*C you can obtain >95% cleavage when you use 1:100 ratio protease to
substrate. Increasing the temperature speeds up the cleavage dramatically and
at room temperature, you can obtain the same efficiency for 10 mins approx.
I am assuming you are interested in using the 3C protease to cleave a tag from
recombinantly expressed protein.
Kind regards,
Nikolay
On 02/03/2022 6:25 PM Srivastava, Dhiraj <[email protected]> wrote:
Hi
sorry for off topic question. does anyone know which protease inhibitors we
can include safely while cleaving with 3C protease?
Thank you
Dhiraj
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Nikolay Dobrev
Postdoctoral Fellow @ Wilmanns group
EMBL Hamburg, c/o DESY, Building 25A,
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