 |
 |
Hi Tony, You may want to start by doing limited proteolysis and running gels on the samples to see if there are any obvious bits that can be cloned out. We generally have the protein of interest at 1 mg/mL and add protease at a 1:200 ratio. At various time points (5, 20, 60, 120, 240 minutes, overnight) take out an aliquot and stop the proteolysis (I just put it in 5X loading dye and boil it). Mass spec the bands of interest and you should be good to go. Of course, if you're cutting out an internal loop you'll end up with two bands, but the hope is to find something consistent among the different proteases. Regards, John On Fri, Oct 30, 2015 at 7:19 AM, Antonio Ariza <[log in to unmask]> wrote: > Hi all, > > > > I have a couple of proteins that are stable as full-length constructs, but > have refused to crystallise after many attempts with different buffers and > ligands. I'd like to try *in-situ proteolysis *with these before I start > cloning truncated versions of them. > > > > Have any of you used this method and do you have any tips? > > > > Cheers, > > > > Tony > > > ------------------------------------------------------ > > > > > > > * Dr. Antonio Ariza University of Oxford Sir William Dunn School of > Pathology South Parks Road Oxford OX1 3RE* > ------------------------------ > *From:* CCP4 bulletin board [[log in to unmask]] on behalf of Eleanor > Dodson [[log in to unmask]] > *Sent:* 30 October 2015 07:37 > *To:* [log in to unmask] > *Subject:* Re: [ccp4bb] Fo-Fo map creation > > Well - it is easy in the GUI > Use cad (reflection utils) to get an mtz file with F_LIg .. F_Papo-- > PHICapo FOM_apo > > Then scale it to make sure the Fapo and FLig are on the same scale. > The analysis of Riso gives some idea of isomorphism > > The Map utilities > > F1 = FLig F2 = F_apo PHI = PHIC W = FOM > > That will give a quick picture, similar to that generated by DIMPLE - > there are more sophisticated procedures but this should be enough to check > if the Ligand IS there. > > Eleanor > > On 29 October 2015 at 19:55, Hena Dutta <[log in to unmask]> wrote: > >> Dear CCP4BB Members, >> I am trying to create Fo-Fo map from two data sets. >> 1. FP_apo, SIGFP_apo >> cell: 160.91, 70.73, 110.51, 90, 130.65, 90; space gr. C2 >> & >> 2. F_LigBound, SIGF_LigBound >> cell: 161.03, 69.74, 112.59, 90, 130.31, 90; space gr C2 >> >> I have good solution for the receptor with both data sets using PHASER >> MR. >> >> Phenix could not work as the cell dimensions are not close enough. >> Changing some criteria in phenix may work. But, I don't know how to do that. >> >> Can someone tell me the steps in CCP4? >> >> I don't have IMEAN in my mtz file, so could not use DIMPLE. I did not >> find a way to create IMEAN from F in CCP4. Sorry, if my question is trivial. >> >> Best regards, >> Hena >> >> >> >> >> >