Ideally, the more nonspecific the protease, the better. (That would be a separate protein engineering project, or chemical catalyst project..) For now, we found that Pronase works well enough. Cheers, Jing
-- Jing Huang, Ph.D. Associate Professor UCLA Department of Molecular & Medical Pharmacology 310-825-4329 jinghuang at mednet dot ucla dot edu http://labs.pharmacology.ucla.edu/huanglab/ ________________________________________ From: Jacob Keller [[email protected]] Sent: Monday, September 05, 2011 3:44 PM To: Huang, Jing Cc: [email protected] Subject: Re: [ccp4bb] Trying to "digest" PISA results Good ol' trypsin--any reason why not? JPK On Mon, Sep 5, 2011 at 5:40 PM, Huang, Jing <[email protected]> wrote: > Excellent point indeed. We always include (at least one, preferably multiple) > control proteins that are proteolysed equally across to make sure that the > observed "target stabilization" is not due to fortuitous protease inhibition. > > What protease did you use for your nucleotide binding case? The protease > sometimes matters. For example, thermolysin mainly only digests proteins that > are unfolded, whereas pronase, which is a mixture of various proteases, can > digest both folded and unfolded proteins. > > Best, > Jing > > -- > Jing Huang, Ph.D. > Associate Professor > UCLA > Department of Molecular & Medical Pharmacology > 310-825-4329 > jinghuang at mednet dot ucla dot edu > http://labs.pharmacology.ucla.edu/huanglab/ > > ________________________________________ > From: Jacob Keller [[email protected]] > Sent: Monday, September 05, 2011 12:18 PM > To: Huang, Jing > Cc: [email protected] > Subject: Re: [ccp4bb] Trying to "digest" PISA results > > I did a similar assay years ago, but since the results were negative, > never published anything--it was seeing whether nucleotides bound to > my protein of interest by time courses of proteolysis +/- nucleotide. > One tricky part of the assay, however, is to be sure that the compound > of interest doesn't inhibit the protease--did you address that? I > guess you would have to have some control proteins for that... > > Jacob > > > g >> >> -- >> Jing Huang, Ph.D. >> Associate Professor >> UCLA >> Department of Molecular & Medical Pharmacology >> 310-825-4329 >> jinghuang at mednet dot ucla dot edu >> http://labs.pharmacology.ucla.edu/huanglab/ >> >> ________________________________________ >> From: CCP4 bulletin board [[email protected]] On Behalf Of Jacob Keller >> [[email protected]] >> Sent: Monday, September 05, 2011 10:10 AM >> To: [email protected] >> Subject: Re: [ccp4bb] Trying to "digest" PISA results >> >> NMR...take that! >> >> JPK >> >> 2011/9/5 Andreas Förster <[email protected]>: >>> AUC ! >>> >>> >>> Andreas >>> >>> >>> >>> On 05/09/2011 6:00, Jacob Keller wrote: >>>> >>>> mea culpa! How about FRET? >>>> >>>> JPK >>>> >>>> On Mon, Sep 5, 2011 at 11:39 AM, Bosch, Juergen<[email protected]> wrote: >>>>> >>>>> Hi Jacob, >>>>> you forgot cross-linking to stabilize a weak complex and verify that it >>>>> exists. >>>>> Jürgen >>>>> On Sep 5, 2011, at 11:45 AM, Jacob Keller wrote: >>>>> >>>>> Well, I guess I have always been curious what is the gold standard >>>>> here--perhaps SEC, ITC, SPR, pulldowns? What if SEC shows a >>>>> polydisperse sample with weak oligomerization, or SPR a very weak >>>>> binding constant? Do we then revert to a functional assay? Or what if >>>>> the functional assay does not show anything, but the binding constant >>>>> is really strong? Or vice versa, the binding is completely >>>>> undetectable, but the functional assay shows something? >>>>> >>>>> JPK >>>>> >>> >>> >>> -- >>> Andreas Förster, Research Associate >>> Paul Freemont & Xiaodong Zhang Labs >>> Department of Biochemistry, Imperial College London >>> http://www.msf.bio.ic.ac.uk >>> >> >> >> >> -- >> ******************************************* >> Jacob Pearson Keller >> Northwestern University >> Medical Scientist Training Program >> cel: 773.608.9185 >> email: [email protected] >> ******************************************* >> >> IMPORTANT WARNING: This email (and any attachments) is only intended for >> the use of the person or entity to which it is addressed, and may contain >> information that is privileged and confidential. You, the recipient, are >> obligated to maintain it in a safe, secure and confidential manner. >> Unauthorized redisclosure or failure to maintain confidentiality may subject >> you to federal and state penalties. If you are not the intended recipient, >> please immediately notify us by return email, and delete this message from >> your computer. >> > > > > -- > ******************************************* > Jacob Pearson Keller > Northwestern University > Medical Scientist Training Program > cel: 773.608.9185 > email: [email protected] > ******************************************* > > IMPORTANT WARNING: This email (and any attachments) is only intended for the > use of the person or entity to which it is addressed, and may contain > information that is privileged and confidential. You, the recipient, are > obligated to maintain it in a safe, secure and confidential manner. > Unauthorized redisclosure or failure to maintain confidentiality may subject > you to federal and state penalties. If you are not the intended recipient, > please immediately notify us by return email, and delete this message from > your computer. > -- ******************************************* Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: [email protected] ******************************************* IMPORTANT WARNING: This email (and any attachments) is only intended for the use of the person or entity to which it is addressed, and may contain information that is privileged and confidential. You, the recipient, are obligated to maintain it in a safe, secure and confidential manner. Unauthorized redisclosure or failure to maintain confidentiality may subject you to federal and state penalties. If you are not the intended recipient, please immediately notify us by return email, and delete this message from your computer.
