Hello All,
Does anyone know of an agent that will break up a protein-protein
interaction without eluting an MBP-tagged protein from amylose resin? I am
trying to do a protein-protein binding assay with an immobilized MBP-tagged
protein. Granted, the putative binder will be easy to distinguish on
SDS-PAGE, so I could simply elute the whole mess with SDS, maltose, urea,
etc., but it would be cleaner to just knock off the binding protein (I plan
to use a lot of the MBP-tagged protein, so the gel would get a big
distorting protein blob). Also, we do not have a good antibody for the
putative binding protein, so a western is out of the running, although we do
have tons of both proteins, hence coomassie is IN the running.
Thanks,
Jacob Keller
*******************************************
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
Dallos Laboratory
F. Searle 1-240
2240 Campus Drive
Evanston IL 60208
lab: 847.491.2438
cel: 773.608.9185
email: [log in to unmask]
*******************************************
----- Original Message -----
From: "Sabine Schneider" <[log in to unmask]>
To: <[log in to unmask]>
Sent: Tuesday, September 16, 2008 11:39 AM
Subject: [ccp4bb] difference in data processed with xscale and scala
> Hello everyone,
>
> I am puzzled about differences I see when I refine the very same structure
> against data processed with xscale or scala.
>
> I got data to 2.55A from a protein-ligand complex. The data were processed
> with xds/xscale (1) or xds/scala (2)
> Free R was imported from a previously solved structure with a different
> ligand, first to (1) and from there to (2).
> After MR and refinement (Refmac/Phenix), I get some difference density
> peaks (a bit pos and neg), near the ligand when I refine against (1) and
> it converges more or less around R/Rfree of 20.9 and 24.8. I do not get
> these pos and negative density blobs when I refine the same coordinates
> against (2), with a tiny bit higher R/Rfree of 21.6/26.1?
> So the data processed with scala, where I get slightly better refinement
> statistics, I end up with some pos/neg density peaks. They don't show up
> when I apply a high resolution cut-off of 2.65A during refinement.
>
> Symmetry is orthorhombic and the statistics are OK (Rsym 0.09 and 0.38 in
> highest shell, redundancy ~5, I/sigI =1.9, mean I/sigI = 5.2 ) and in
> both files appear to be more or less the same number of reflections.
>
> I also did simulated annealing, omitting the region around the ligand in a
> 15A radius and the density is nicely coming up. But it doesn't make a
> difference: after refinement I always end up with the result as discribed
> above? And there isn't really a difference between the two structures in
> the end.
>
> Has anyone an idea whats going on?
>
> Sabine
>
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