Fluorescent stains for gels

This document describes current knowledge and experience in the BRF of fluorescent protein stains for gels.

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

In the case of Coomassie, the disadvantages of fluorescent stains may be outweighed by the greater sensitivity. For silver staining, the lower reagent cost may not worth the extra effort required to silver stain, and the limited 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.

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. A disadvantage of  Sypro Ruby is the presence of speckles cause by precipitation of the dye.

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.

An alternative to Sypro Ruby is Deep Purple stain from GE HealthCare. 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.

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. 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

There is a fluorescent stain for glycoproteins, using traditional chemistry, but with a fluorescent dye attached. Note that there are two version of the stain, one requiring illumination with UV light, the other with visible light. The former can be seen on a aUV 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 from transmembrane proteins. They claim that unlike some older stains, it stains transmembrane proteins rather than hydrophobic proteins.

Visualizing fluorescently stained gels

One of the appeals of gels has been the ability to see bands and spots, an appeal which is lost with fluorescent stains. With some stains e.g. Sypro Ruby, the gel can be examined visually on a UV transilluminator, using protection from UV light for the viewer. However, a number of stains can only be seen with a fluorescent scanner. To cut spots of interest from a fluorescently stained gel also requires a machine.

Equipment in the Biomolecular Research Facility

The BRF has a Bio-Rad FX scanner equipped with the standard 532 nm laser and the optional 488 nm and 635 nm lasers. A selection of filters allows us to see numerous common stains.

Bio-Rad PDQuest software controls the instrument. Data files are stored in a proprietary format, but can be converted to 8 or 16 bit TIFF files. Images from scans can be overlaid with a program like Photoshop

Data can be transferred with a USB flash drive, email, CD or Zip disk.

The BRF also has a spot cutter which takes a low resolution image with a 300 nm light, suitable for Sypro ruby (the machine can also detect visible stains). Spots selected on the image are then cut by the instrument, which deposits gel plugs in a 96 well plate.