Written and maintained by CASRAI Editorial Board
Last updated
Who this is for: bench researchers choosing a protein gel stain, or wondering why a thermal shift protocol calls for a dye with the same name. After reading you should know what SYPRO Ruby and SYPRO Orange are, how they differ, and which questions to settle before staining a gel. This is general reference information, not a validated protocol.
Short answer
SYPRO is a family of fluorescent dyes from Molecular Probes (Thermo Fisher). SYPRO Ruby is a ready-to-use, ultrasensitive luminescent stain for proteins in one- and two-dimensional polyacrylamide gels, with a lower detection limit of about 0.25 to 1 ng. SYPRO Orange is a separate dye supplied as a 5,000x concentrate in DMSO, used for SDS-PAGE gels and, in a different role, as the reporter dye in thermal shift assays.
Definition and scope
The name “SYPRO” covers several distinct products, so a protocol that says only “SYPRO” is ambiguous. The two covered here are those with a manufacturer document read for this page.
- SYPRO Ruby protein gel stain is described by its manufacturer as a ready-to-use, ultrasensitive, luminescent stain for proteins separated by PAGE, with performance “as good as or better than the best silver staining techniques.” It is supplied as a 1X solution (200 mL, 1 L and 5 L pack sizes are listed in the product information).
- SYPRO Orange protein gel stain is a fluorescent stain for SDS or native polyacrylamide gels, supplied as a 5,000x concentrate in DMSO and stored at 4 °C (Bio-Rad instruction manual, cat. 170-3120).
How SYPRO Ruby is used in practice
According to the Molecular Probes product information, SYPRO Ruby has excitation maxima near 280 and 450 nm and emission near 610 nm. It binds basic amino acids and the polypeptide backbone, which the manufacturer gives as the reason for less protein-to-protein variability than silver staining. It stains most protein classes, including glycoproteins, phosphoproteins and lipoproteins, and does not stain extraneous nucleic acids. Quantitation is linear over about three orders of magnitude.
The manufacturer describes two workflows at overview level. The basic protocol runs overnight (about 18 hours in total for a 1 mm minigel), gives sensitivity near 1 ng and the widest linear range. The rapid protocol uses a microwave, takes about 90 minutes, has a detection limit of 0.25 to 1 ng, and was optimized for NuPAGE minigels. Both involve fixing the gel, staining, a short wash and a water rinse before imaging. Exact solutions, volumes and times are in the manufacturer’s product information and should be followed from there.
Imaging uses UV transillumination or lasers; the manufacturer lists 473 to 488 nm and 532 nm lasers as optimal. Because the stain does not covalently alter the protein, the product information states it is compatible with Edman sequencing and mass spectrometry, and it can be multiplexed with the Pro-Q Diamond (phosphoprotein) and Pro-Q Emerald 300 (glycoprotein) stains.
SYPRO Ruby versus SYPRO Orange
The table sets out only what the two manufacturer documents state. Cells marked “not stated” mean the document read does not give the value.
| Feature | SYPRO Ruby | SYPRO Orange |
|---|---|---|
| Supplied as | Ready-to-use 1X solution | 5,000x concentrate in DMSO |
| Storage | Room temperature, protected from light; stable at least 9 months | 4 °C |
| Excitation | 280 and 450 nm maxima; UV transillumination or 473 to 488 nm and 532 nm lasers | UV (about 302 nm) or 488 nm |
| Gel types | 1-D and 2-D PAGE | SDS or native PAGE |
| Stated sensitivity | 0.25 to 1 ng lower limit | Not stated |
| SDS in the staining solution | Not stated | Must be absent (high background); native gels are soaked in SDS first |
| Container | Polypropylene or polycarbonate; glass not recommended | Plastic trays |
Compared with other stains already covered on this site, Coomassie Blue is a visible-light dye, whereas both SYPRO stains are fluorescent and need an appropriate imager. For gel setup, see SDS-PAGE: how protein gel electrophoresis works.
SYPRO Orange in thermal shift assays
SYPRO Orange is also the dye most often used in thermal shift assays (differential scanning fluorimetry). There it is added to purified protein in solution and fluoresces more as heating exposes hydrophobic regions, which is a different use from post-run gel staining. The mechanism and curve reading are covered in Thermal Shift Assay (DSF). The gel-staining and thermal-shift protocols are not interchangeable.
Where it matters for labs: a pre-staining checklist
These questions follow directly from the manufacturer documents and help decide whether SYPRO Ruby fits a given gel.
- Time or linear range? Overnight basic protocol for the widest linear range; rapid protocol for a 90-minute turnaround.
- Can the imager excite and read it? Ruby needs UV or a 473 to 488 nm or 532 nm source; check the emission filter against the roughly 610 nm emission.
- Downstream analysis? Mass spectrometry and Edman sequencing are stated as compatible.
- Multiplexing? Phosphoprotein and glycoprotein stains from the same product family can be combined on one gel.
- Containers and light? Use plastic, not glass, and cover the tray to protect from light.
Handling and waste: the manufacturer characterizes the stain as an irritant because of its solvent and buffer salts, says the ruthenium component is not a regulated hazardous heavy metal in the US, and recommends that used stain be combined with flammable waste for incineration rather than poured down the drain. Local rules may be stricter, so follow your institution’s chemical waste policy and the safety data sheet. Ladders run alongside stained samples are covered in the Precision Plus Protein Ladder guide.
Common misunderstandings
- “SYPRO” is one product. It is a family; Ruby and Orange differ in format, excitation and use.
- Longer staining always means better results. The manufacturer says gels can sit in SYPRO Ruby indefinitely without overstaining, but speckling artifacts increase.
- The thermal shift dye and the gel stain are used the same way. They share a name and a dye family, not a workflow.
FAQ
What is SYPRO Ruby?
An ultrasensitive, ready-to-use luminescent stain for proteins in 1-D and 2-D polyacrylamide gels, made by Molecular Probes (Thermo Fisher).
How sensitive is SYPRO Ruby?
The manufacturer states a lower detection limit of 0.25 to 1 ng of protein, with linear quantitation over about three orders of magnitude.
Is SYPRO Ruby compatible with mass spectrometry?
Yes. The manufacturer states compatibility with mass spectrometry and Edman sequencing.
What is the difference between SYPRO Ruby and SYPRO Orange?
Ruby is a ready-to-use 1X gel stain; Orange is a 5,000x DMSO concentrate used for SDS or native gels and also as the reporter dye in thermal shift assays. See the table above.
Sources
- Molecular Probes / Thermo Fisher Scientific, SYPRO Ruby protein gel stain, Product Information MP 12000 (rev. 20 Nov 2007), documents.thermofisher.com, read 2026-11-07.
- Bio-Rad Laboratories, SYPRO Orange Protein Gel Stain Instruction Manual, cat. 170-3120, bio-rad.com, read 2026-11-07.
- CASRAI guides on Thermal Shift Assay, Coomassie Blue and SDS-PAGE, linked above.
