Protein Protocols Protein Protocols

284 Patton
filter. Proteins stained with the dye can also be visualized using imaging systems equipped
with 450-, 473-, 488-, or even 532-nm lasers.
4.5. Luminescent Detection of Proteins in SDS-Polyacrylamide Gels
Using SYPRO Ruby Protein Gel Stain
14. Polypropylene dishes, such a Rubbermaid® Servin’ Savers, are the optimal containers for
staining because the high-density plastic adsorbs only a minimal amount of the dye. Clean
and rinse the staining containers well before use, as detergent will interfere with staining.
It is best to rinse the containers with ethanol before use. For small gels, circular staining
dishes provide the best fluid dynamics on orbital shakers, resulting in less dye aggregation
and better staining. For large format 2-D gels, polyvinyl chloride photographic staining
trays, such as Photoquip Cesco-Lite 8 in. × 10 in. photographic trays also work well. Glass
dishes are not recommended.
15. Minimal staining volumes for typical gel sizes are as follows:
50 mL, for 8 cm × 10 cm × 0.75 mm gels (minigels)
330 mL, for 16 cm × 20 cm × 1 mm gels
500 mL, for 20 cm × 20 cm × 1 mm gels
or ~10 times the volume of the gel for other gel sizes
16. Use only fresh staining solution for optimal sensitivity. Longer staining times result in
greater sensitivity. Using too little stain will lower the sensitivity.
17. Always store gels in the dark to prevent photobleaching. When gels are stored in the staining
solution, the signal decreases somewhat after several days; however, depending on the
amount of protein in bands of interest, gels may retain a usable signal for many weeks.
18. The stained gel is best viewed on a standard 302 nm UV or a blue-light transilluminator.
Gels may also be visualized using various laser scanners: 473 nm (SHG) laser, 488 nm
argon-ion laser, 532 nm (YAG) laser. Alternatively, use a xeon arc lamp, blue fluorescent
light, or blue light-emitting diode (LED) source. Gels may be photographed by Polaroid
or CCD camera. Use Polaroid 667 black-and-white print film and the SYPRO protein gel
stain photographic filter (Molecular Probes, Inc., cat. no. S-6656). Exposure times vary
with the intensity of the illumination source; for an f-stop of 4.5, roughly 1–3 s should be
required.
4.6. Luminescent Detection of Proteins in IEF Gels
Using SYPRO Ruby IEF Protein Gel Stain
19. Minimal staining volumes for typical gel sizes are as follows:
50 mL, for 6 cm × 9 cm × 1 mm gels (minigels)
150 mL, for 22 cm × 22 cm × 1 mm (Multiphor II format gels, Amersham-Pharmacia
Biotech)
0r approx 10 times the volume of the gel for other gel sizes.
20. Polypropylene dishes, such as Rubbermaid ® Servin’ Savers, are the optimal containers for
staining because the high-density plastic adsorbs only a minimal amount of the dye. Clean
and rinse the staining containers well before use as detergent will interfere with staining.
It is best to rinse the containers with ethanol before use. For small gels, circular staining
dishes provide the best fluid dynamics on orbital shakers, resulting in less dye aggregation
and better staining. For large-format 2-D gels, polyvinyl chloride photographic staining
trays, such as Photoquip Cesco-Lite 8 in. × 10 in. photographic trays also work well. Glass
dishes are not recommended.
21. The stained gel is best viewed on a standard 302-nm UV or a blue-light transilluminator.
Gels may also be visualized using various laser scanners: 473-nm (SHG) laser, 488 nm