Good ol' trypsin--any reason why not? JPK
On Mon, Sep 5, 2011 at 5:40 PM, Huang, Jing <jinghu...@mednet.ucla.edu> 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 [j-kell...@fsm.northwestern.edu] > Sent: Monday, September 05, 2011 12:18 PM > To: Huang, Jing > Cc: CCP4BB@JISCMAIL.AC.UK > 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 [CCP4BB@JISCMAIL.AC.UK] On Behalf Of Jacob Keller >> [j-kell...@fsm.northwestern.edu] >> Sent: Monday, September 05, 2011 10:10 AM >> To: CCP4BB@JISCMAIL.AC.UK >> Subject: Re: [ccp4bb] Trying to "digest" PISA results >> >> NMR...take that! >> >> JPK >> >> 2011/9/5 Andreas Förster <docandr...@gmail.com>: >>> 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<jubo...@jhsph.edu> 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: j-kell...@northwestern.edu >> ******************************************* >> >> 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: j-kell...@northwestern.edu > ******************************************* > > 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: j-kell...@northwestern.edu *******************************************
