Gel scanning, fluorescent stains, spot picking
The Biomolecular Research Facility has equipment which may be useful for those running 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. This is a self-service instrument
For common fluorescent stains, the
facility has a Bio-Rad FX fluorescent scanner operated by the core (fee
for service). Stains we have scanned for are Sypro Ruby, Pro-Q Diamond
phosphoprotein 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 wavelength for emission
readings, enabling many common dyes to be scanned.
Bio-Rad has a list of fluorescent stains that the FX scanner detects.
Although not tested, the scanner should 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 of 2-D gels can be
analyzed with Bio-Rad PDQuest software, a time consuming
process because of streaks and faint spots amidst other factors.
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 UV-B light, but there is no wavelength selection. For fluorescent stains, there is a 620/60 nm bandpass filter installed, this system was optimized 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 .
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.
Advantages of fluorescent stains
Disadvantages of fluorescent stain
greater dynamic range
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 using 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.
Pro-Q 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 phosphorylated 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
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.
Molecular Probes sell a stain selective for transmembrane proteins. They claim that unlike some older stains, it stains transmembrane proteins rather than hydrophobic proteins.