Dear Dhiraj,
yes, on-column cleavage works as well quite efficiently with 3C protease.
I am performing it in a cold room (8-10C) for 1h normally reaches similar
efficiency.
Depending on your tags (if the 3C has a His-tag) you can add even more protease
1:50 then for sure 1h is more than enough.
The only thing you need to ensure is that upon cleavage your protein does not
bind to the IMAC resin due to internal Histidines. If that is the case you can,
just collect the flow-through and pass it over the beads couple of more times
to ensure efficient binding of the His-3C protease then you should have a
highly pure target.
Please note, I am referring to Ni-NTA Agarose beads, which you use in bach mode.
The efficiency will be lower most like if you are using pre-packed columns.
I hope that is helpfull.
Best, Nikolay
> On 02/03/2022 6:56 PM Srivastava, Dhiraj
> wrote:
>
>
> 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 on behalf of Nikolay
> Dobrev
> Sent: Thursday, February 3, 2022 11:53 AM
> To: CCP4BB@JISCMAIL.AC.UK
> 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
> 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
> >
> >
> > -
> >
> > To unsubscribe from the CCP4BB list, click the following link:
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> >
> > >
> Nikolay Dobrev
> Postdoctoral Fellow @ Wilmanns group
> EMBL Hamburg, c/o DESY, Building 25A,
> Notkestraße 85, 22607 Hamburg, Germany
> T +49 40 89902 165 | M +49 173 684 0532
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>
>
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Nikolay Dobrev
Postdoctoral Fellow @ Wilmanns group
EMBL Hamburg, c/o DESY, Building 25A,
Notkestraße 85, 22607 Hamburg, Germany
T +49 40 89902 165 | M +49 173 684 0532
twitter.com/emblevents https://twitter.com/emblevents
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Visit http://www.embl.org/events for a complete list of all EMBL events.
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