SYPRO-Orange

Advantages: Simple, quick staining protocol As sensitive as silver staining (~2ng/band for most proteins) Linear range from 2ng to at least 1 microgram Allows western transfer and immunodetection after staining Nonspecific fluorescent staining of denatured proteins in 1D and 2D SDS-polyacrylamide gels Excitation Maximum: 300 nm Emission Maximum: 570 nm Binding: Nonspecific, SDS-dependent binding to proteins Detection Limits: As for all protein staining systems, dye binding will depend on the amino acid composition of the proteins. Proteins in native gels will exhibit more staining variability than proteins saturated with SDS.

56ng/band for most proteins in denaturing gels

Run gel.
Dilute SYPRO Orange 1:5000 in 7.5% acetic acid (enough to cover gel).
Shake vigorously.
Place gel and stain in a foil-covered container, and shake gently for 30-60 min.
Optional: destain in 7.5% acetic acid for 10-15 min. Rinse briefly in distilled water.

Alternative Protocol for Western Transfer:
Dilute SYPRO Orange 1:5000 in Tris/Glycine/Methanol transfer buffer. Proceed with staining as above.
If necessary, destain in transfer buffer.

Linear Range: 2 orders of magnitude.

Emission Filters: SYPRO-Orange filter

Storage: Stock solution is stable for six months to one year stored at -20 degrees C and protected from light.
Staining reagent diluted in buffer or 7.5% acetic acid can be stored protected from light at 4 degrees C for
at least three months.

Handling: No toxicity data available. Stock solution contains DMSO and should be handled with double gloves.

Vendor: Molecular Probes, Inc. www.probes.com

Useful tips for SYPRO-Orange

Ensure glass plates and staining containers are completely free of grease and fingerprints.
Use powder-free gloves to handle gels.
Mix staining solution fresh immediately before using.
Shake staining solution vigorously before adding it to the gel.
Staining in acetic acid significantly impairs immunodetection.
For western blotting, stain in transfer buffer (sensitivity may be slightly diminished.)
Staining solution can be reused effectively at least three times.
When staining native gels, soak gels in 0.1% SDS (in either 7.5% acetic acid or water)
for 30-60 min prior to staining.
Sensitivity will be diminished in native gels.
Methanol can adversely affect staining.
To eliminate methanol, soak gel in 7.5% acetic acid for 1-2 days prior to staining.
When staining acidic proteins, increase the concentration of SDS in the gel and buffers to 0.2%.