Hello there,
I have successfully used Sypro Ruby on blot for quality control before Western
detection using chemiluminescence. So here are a couple of thoughts and remarks:
- Sypro Ruby is quite easy to use but not that sensitive. It is slightly less
sensitive than amido black on membrane (which is actually not that bad). I think
it less sensitive than Sypro Ruby for gel (probably because you have a membrane
that diffuses out the emitted light).
- We have used it on nitrocellulose and on PVDF. The membrane on choice will
depend on your antibody.
- We have not found Sypro Ruby to interfere with the Western signal. We used
PVP-40 as blocking reagent instead of milk, BSA or others because we wanted to do
colloidal gold staining after the immunoreaction (a protein blocking reagent will
cause a very heavy background). Using PVP-40 as blocking agent, we have
demonstrated that the Sypro Ruby stain is washed from the protein (it is a
non-covalent interraction) and hence will not interfere with the immunoreaction.
- We have used a CCD camera to record the pattern. A hand-held UV light will
obviously also do the job although in a quite less quantitative manner. However,
the main reason why we used the CCD camera is that we can also record the
chemiluminescence signal from the Western on the same camera. If you have
recorded the two images on the same scale, you can immediately match the two
images to each other and you can therefore easily locate the band/spot that was
immunoreactive on the blot.
- The method development has been described in the following reference: Ducret A.
et al. (2000) Electrophoresis 21 pp 2196-2208.
Hope that this helps
Axel
Di Barraclough wrote:
> HI there,
> I'm thinking of using the Sypro Ruby stain for membrane blots.
> Is there anyone out there who also uses this product?
> If so, I have three main questions of you:
> 1) what do you use for EPI-illumination nad how do you document the
> image? (Do you use handheld UV lamps from above, or something more
> sophisticated?)
> 2) does staining with Sypro interfere with wanting to perform subsequent
> antibody work with the membrane?
> 3) Are there any other issues that you found were important?
>
> Regards
> Di Barraclough.
>
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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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