Dear Pascal and Toufic, Many thanks to both of you for the pointers. Toufic, I actually poked around online and bought exactly the same kit that you are suggesting, especially because the beads are reusable. So your experience is reassuring. So I'll find out soon.
I also plan to play around with the detergent type and concentration among other things. I have no plans to take the shortcut but it's nice to have at least one other human being to talk to on these matters. I appreciate your responses :) Many thanks! Raji On Wed, Feb 20, 2013 at 1:15 PM, Toufic El Arnaout <elarn...@tcd.ie> wrote: > Hi Raji, > I addition to the tips from Pascal, I would like to say that for a memb > protein I worked on with a his-tag separated by a thrombin site, I used > thrombin cross linked to agarose from Sigma (1 mL). The beads can be > collected, washed and reequilibrated, making it ready for use so many times. > In my case 1 mL of this thrombin was enough to cleave up to 5-10 mg of > target protein, at 4 °C for 16 h (with slight rotation in a 15 mL tube, > total volume up to 10 mL). In FC-12 there was a little better cleavage than > in DDM. > My opinion is that how thrombin (or most other proteases) will cleave may > mostly depend on your protein/fusion type/protein-micelle complex > structure/access to the site... > You just have to try. Best wishes. > toufic > > > On Wed, Feb 20, 2013 at 5:15 PM, Raji Edayathumangalam > <r...@brandeis.edu>wrote: > >> Hi Folks, >> >> Sorry this isn't a non-ccp4 post. >> >> I am working with a membrane protein for which I am finally able to scale >> up expression. I am now also able to partially purify my protein from a >> medium-scale (12-18L) bacterial culture using a two-step tandem affinity >> purification protocol (Talon followed by amylose affinity steps). As the >> next purification step, I am about to set up a pilot thrombin cleavage >> experiment to separate my protein from the His.MBP fusion tag (see below). >> >> The construct that I am working with is as follows: >> His.MBP--ThrombinSite--Membrane Protein >> >> There is only one theoretical thrombin cleavage site in the entire fusion >> protein i.e., at the desired cleavage site with no theoretical secondary >> sites. I would like to try cleavage both at 4C and around 25C from 4h to >> overnight but I also have to balance the trials with the material I must >> generate for the endless permutations and combinations one can try. Each >> sensible pilot experiment is going to use up partially purified protein >> from 6-12L preps. >> >> FYI. All purification buffers contain DDM and I haven't yet done >> extensive detergent screens. >> >> Please could I ask the community to share tips/suggestions about >> large-scale thrombin cleavage experiments with their favorite membrane >> proteins. >> >> Many thanks. >> Raji >> >> >> -- >> Raji Edayathumangalam >> Instructor in Neurology, Harvard Medical School >> Research Associate, Brigham and Women's Hospital >> Visiting Research Scholar, Brandeis University >> >> > > > -- > ****************************************************** > Toufic El Arnaout > Trinity Biomedical Science Institute (TCD) > 152-160 Pearse Street, Dublin 2 > Tel.: +353 85 83 40 157 > ****************************************************** > -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
