David,
I would first add another camera manufacterer, Fuji Medical, with their camera L
AS-1000S. In our hands it proved better than the
BioRad system. Its sensitivity is comparable to X-Ray film and we have very good
linearity. We are actually looking at how we can
get better quantitation of Western-detected 2-D spot, a really not trivial probl
em as many of you may know. One of the trick that we
are learning right now is that, in contrast to X-Ray film, it pays off to expose
longer. A typical blot will yield a decent pattern
with a 5 min exposure. However, the signal linearity (not the signal strength)
will dramatically increase if we expose for 30 min,
so that the spot recognition by PDQuest or Melanie (we use the latter) will be s
implified (in particular, the background will be
more homogeneous so that the signal to noise ratio is improved).
A direct consequence of a long exposure is a need for a 16-bits capable camera.
Because you have so many grey level available you
can expose longer without saturating the camera and, therefore, obtain images of
better quality. We have tried to analyze with
Melanie 16-bits 2-D blots image in 16-bits mode, in 12-bits mode (conversion per
fomed by Melanie) and in 8-bits mode (the mode
change was performed first with Photoshop). The 12-bits mode seems to work the b
est, probably because the algoryhm provided by
GeneBio is optimized for a 12-bits image. I got almost similar results with the
16-bits image after a number of changes in the
detection parameters. The 8-bits image was definitely noiser and weaker spot did
not seem to be detected properly.
And just in case somebody was wondering: I am not affiliated with Fuji Medical i
n any way, but I am just a happy user of one of
their camera.
Axel
David Dogruel wrote:
> We are also dealing with some of the issues that Alex Bell
> asked about and Katheryn Resing commented on regarding
> 2-D imaging and image analysis. Our gels are now
> fairly reproducible and give very nice patterns with silver
> staining. We have also begun to identify some of the
> stronger spots with MALDI peptide mapping, and hope
> to increase our success rate with low-flow LC and
> ESI on the LCQ.
>
> We will be evaluating the SYPRO stains at a later date, but
> the reality of today is silver and coomassie stains.
> So, now it's time to image the gels and pull out differences
> in spot patterns.
>
> There appear to be 4 major imagers that can handle _both_
> colorimetric stains (silver, coomassie) and the fluorescent
> protein stains (SYPRO) - the Bio-Rad Fluor-S or Fluor-S Max,
> the Hitachi CDBIO 8C or 16C, the Genomic Solutions Investigator Proteomic
> Analyzer Camera-based system, and the Alpha Innotec
> Fluor-Chem. The Pharmacia system appears to be undergoing
> some revisions/improvements and I couldn't find enough
> info about it for comparison purposes.
> We are not doing any rad work, so the phosphor imagers,
> while nice, are not going to be useful for us.
> Anyone have any experience with these imagers
> that they would be willing to share? We have another
> Fluor-S in the building, and the folks who use it for
> lane-based DNA gels really like it.
>
> Katheryn brought up a good point about the 8 bit limit of
> some of the software programs. But if, as she suggests,
> there may be major innovation in 2-D analysis software
> soon, that limit may be overcome and a higher bit image
> may become necessary. So one question is whether it
> is a good hedge bet to go for a 16 bit camera system now
> (vs. a 12 bit camera system) to avoid running into a resolution
> limit in the future.
>
> We are also looking at image analysis programs, and
> one that wasn't mentioned previously is the new version
> of Image Master 2D Elite from Pharmacia. It looks like
> it would fit somewhere between PDQuest and Melanie
> and I am trying to get my hands on a demo.
> Anyone tried this package?
>
> Any info is appreciated.
>
> -Dave
>
> ************************************
> David Dogruel
> Los Alamos National Laboratory
> Bioscience Division
> MS M888
> Los Alamos, NM 87545
>
> Ph: 505-665-9071/8034
> FAX: 505-665-3024
>
> dogruel@lanl.gov
> ***********************************
-- ------------------------------------------------------------------------------ ------------------------------------------------------Axel Ducret, Ph.D. Senior Research Biologist Merck-Frosst Canada Inc. Dept. Biochemistry and Molecular Biology P.O. Box 1005 Pointe-Claire-Dorval PQ H9R 4P8 Canada
tel. + (514) 428-3428 fax + (514) 428-4900
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