University of Virginia Health System

Gel scanning, fluorescent stains, spot cutting

The Biomolecular Research Facility has equipment which may be useful for those running 2-D gels but no longer performs 2-D electrophoresis as a service.

The facility has a Bio-Rad GS-800 scanner for Coomassie and silver stained gels,  blots and films.

For common fluorescent stains, the facility has a Bio-Rad FX fluorescent scanner . Stains we have scanned for are Sypro Ruby, Pro-Q Diamond phospho-protein stain, Cy3, Cy5, Pro-Q Emerald, pyrene labeled compounds.  Our instrument has lasers with wavelengths of 488 nm, 532 nm, 635 nm, and filters control the wavelenght for emission readings, enabling many common dyes to be scanned, as listed by Bio-Rad. Although not yet tested, the scanner is expected to give images of Western blots using fluorescently labeled secondary antibodies, such as ECL  Plex ™ or DyLight with the advantage of greater linearity than with conventional film.

The image files from both scanners can be converted to TIFF format for export. Images can be analyzed with Bio-Rad PDQuest software, a  time consuming process because of streaks and faint spots amidst other factors. We also have Quantity One software from Bio-Rad for analyzing 1-D gels.

spot cutting from gels or PVDF

A spot cutter can cut out spots and lanes, from gels as an alternative to manual cutting. For compatible fluorescent dyes, the spot cutter may be more convenient than manual cutting on a UV light box.

 For large numbers of spots, the spot cutter may be more convenient than manual cutting.

The gel is illuminated with a visible or ultraviolet light, but there is no wavelength selection; this system works for Sypro ruby stained proteins. Using an image of the gel, spots or bands are selected and then the instrument cuts them. After a spot or band is selected on the computer screen, a 1 or 1.5 mm punch cuts out gel plugs and deposits them in a 96 well plate. To cut bands, a line of several 1 mm plugs is cut .

There is a charge for using the scanners.

Fluorescent stains for gels

Fluorescent protein stains like the Sypro series and Deep Purple are alternatives to Coomassie and silver. Advantages and disadvantages are listed below.

The best known total protein stain is Sypro Ruby from Molecular Probes. This stain may be available from other vendors at a slightly lower price. There are other Sypro stains, which are both less sensitive and less expensive. Some people reuse or dilute Sypro Ruby, although Molecular Probes has data showing that dilution or reuse reduces sensitivity.There are reports of making a stain equivalent to Sypro Ruby, although with less sensitivity; some who have tried their own synthesis have been dissatisfied.

Although the protocols from suppliers suggest only one destain step, we recommend multiple changes of destain solution to avoid having a high background. Note that for the last step, put the gel in water. Some sources advise against usiing latex gloves with Sypro stained gels because the gloves can contribute to the background. An annoyance of  Sypro Ruby is the presence of speckles cause by precipitation of the dye.

An alternative to Sypro Ruby is Deep Purple stain from GE HealthCare (formerly Amersham). This stain is claimed to be a little more sensitive than Sypro Ruby and free of the speckling problem, although our initial experience shows speckles. Bio-Rad sell Flamingo Pink fluorescent stain which is claimed to be more sensitive than Sypro Ruby.

fluorescent stains with interesting specificity

There are some novel fluorescent stains available for which visible stains are not available, or have much less sensitivity.

Molecular Probes is the best known supplier of fluorescent protein stains and sells some in packages, which must be used in the specified order. Suitable molecular weight markers may be included to verify that the stain is detecting the specified type of protein.

Phosphoproteins

ProQ-Diamond stain is very selective for phosphoproteins. Some who have used it find more proteins than expected.The phosphoprotein stains must be used before Sypro Ruby for total protein. Molecular Probes suggest using known proteins to establish a ratio of phosphoprotein stain to total protein stain to distinguish between phosphophorylated proteins and non-phosphorylated proteins.

Investigators here have used this stain to follow relative phosphorylation of a a few target proteins on 1-D gels, and in another project to compare the extent of phosphorylation of two proteins.

Non-specific staining may be reduced by extensive washing. Background papers are:

Steinberg, T.H., Agnew, B.J., Gee, K.R, Leung, W-Y, Goodman, T, Schulenberg, B., Hendrickson, J. Beechem, J.M., Haugland, R.P., Patton, W.F. Proteomics 2003, 3, 1128–1144

Schulenberg, B, Aggeler R, Beechem J.M.. Capaldi, R.A. and Patton, W.F. (2003) J. Biol. Chem. 278 27251-27255

Glycoproteins

Molecular Probes sell a fluorescent stain for glycoproteins, using traditional chemistry, but with a fluorescent dye attached. Note that there are two versions of the stain, one requiring illumination with UV light, the other with visible light. The former can be seen on a a UV transilluminator but not the scanner used by the BRF, while the latter stain can be viewed with the BRF fluorescent scanner.

Transmembrane proteins.

Molecular Probes sell a stain selective for transmembrane proteins. They claim that unlike some older stains, it stains transmembrane proteins rather than hydrophobic proteins.