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] *******************************************
