Since people asked:
http://www.usbweb.com/category.asp?cat=118&id=22293
is my preferred source. Please note that I don't have any relation to USB
whatsoever (but large amounts of money sent to my unnamed Swiss bank
account are always appreciated. The password for the account is BACON).
Artem
> The important bit about Thrombin is to find the right source of it. Some
> suppliers are *much* better than others. I've had no problems with this
> protease in the recent year, but had trouble earlier.
>
> Note that Thrombin leaves 2 amino acids whereas TVMV, TEV, and 3C leave
> one amino acid (or even none if you have the right N-terminus).
>
> Artem
>
>> I'd like to third TeV & second 3C ("PreScission").
>>
>> Both have high specificity, good processivity and I have had a lot of
>> success with 3C.
>>
>> I have _never_ got Thrombin to cut cleanly - but I guess I could have
>> been
>> unlucky...
>>
>> Dave
>>
>> On 05/03/07, Cynthia Kinsland <[EMAIL PROTECTED]> wrote:
>>>
>>> Hi,
>>> I'll second the TEV protease suggestion. We use it routinely because
>>> it
>>> is highly specific and easy to make ourselves (and, therefore, cheap).
>>> We
>>> have never seen it cut non-specifically and, since it is cheap, we just
>>> chuck in a bunch and let it go.
>>>
>>> The Prescission protease is also very specific and also available for
>>> home
>>> preparation (it is the 3C protease...prescission is a marketing name).
>>> I
>>> don't have as much experience with it, but it has behaved for me so far
>>> and
>>> I know that a number of people use it routinely with great success.
>>>
>>> Another nice thing about having the clones around to make your own is
>>> that
>>> you can make the protease with the same tag that you intend to cut off
>>> (say,
>>> His or GST or whatever your favorite is). Then, you can remove cleaved
>>> tag,
>>> uncleaved fusion protein and the protease all in one post-cleavage
>>> step.
>>>
>>> In our case, we almost always have a HisTag (often as part of some
>>> larger
>>> fusion) so our TEV is His-tagged. We have some of the GE pGEX vector
>>> for
>>> Prescission protease, so our 3C clone has GST on it.
>>>
>>> Best of luck,
>>>
>>> Cynthia
>>>
>>> On Mar 2, 2007, at 5:01 AM, Rene Frank wrote:
>>>
>>> Hi,
>>>
>>> A non-ccp4 Q. Sorry.
>>>
>>> I would like to use a cleavable purification tag at the
>>> N-terminus/extracellular end of my membrane protein for purification.
>>> Before
>>> I start, I wonder if someone could recommend a particular protease site
>>> that
>>> I can engineer between the tag and my protein? How about a proprietary
>>> cleavage system such as the PreScission protease (GE Healthcare)? I
>>> would
>>> be grateful to hear success and horror stories in this area.
>>>
>>> Best wishes,
>>>
>>> Rene
>>>
>>> ================================================
>>> Dr R.A.W. Frank, PhD
>>> Royal Commission for the Exhibition of 1851 Research Fellow
>>>
>>> Prof Seth Grant Lab / Genes to Cognition
>>> Wellcome Trust Sanger Institute
>>> Hinxton
>>> Cambridge CB10 1SA
>>>
>>> Work Tel: 0044 (0)1223 834244 ext. 7318
>>> Cell No.: 0044 (0)7870 208280
>>> ===============================================
>>>
>>>
>>>
>>>
>>> ____________________
>>> Cynthia Kinsland, Ph.D.
>>> Cornell University
>>> Protein Facility Director
>>> 607-255-8844
>>>
>>>
>>>
>>>
>>
>>
>> --
>> ---------------------------------------
>> David Briggs, PhD.
>> Father & Crystallographer
>> www.dbriggs.talktalk.net
>> iChat AIM ID: DBassophile
>> ---------------------------------------
>> Anyone who is capable of getting themselves made President should on no
>> account be allowed to do the job. - Douglas Adams
>>
>
