ProteOrange Protein Gel Stain is a fluorescent stain for visualization of proteins in polyacrylamide gels after electrophoresis, an analog of SYPRO™ Orange. Proteins bands stained with ProteOrange can be excited by ultraviolet light at approximately 300 nm or by visible light at approximately 470 nm and detected at 570 nm using an appropriate filter.

ProteOrange Protein Gel Stain has many advantages:

✔ 10 times more sensitive than Coomassie™ brilliant blue (CBB) staining and relatively similar to traditional silver staining (detection limit is 3 ng of protein per band for ProteOrange, 30 ng for CBB, 0.3 ng for silver staining)

✔ A simple and quick staining procedure (less than 1 hour)

Low protein-to-protein variability: the stain interacts with the SDS coat around proteins in the gel, so it gives more consistent staining between different types of proteins compared to CBB staining

Easy visualization with routinely used UV or blue-light transilluminator or laser scanner

Broad linear range of detection: fluorescence intensity is linear with protein concentration over three orders of magnitude 

Selective for proteins: ProteOrange detects proteins down to 6.5 kDa and does not stain nucleic acids or lipopolysaccharides. It also stains glycosylated proteins.

In this article, we demonstrate step-by-step instruction on staining proteins with ProteOrange in polyacrylamide gels after electrophoresis. Detailed protocol for staining proteins with ProteOrange you can also find on our website.

After the electrophoresis has been finished, follow the steps below:

Step 1.

Prepare 25 mL of the staining solution. This volume is sufficient for a standard-size 10x15 cm gel.

Prepare 7.5% acetic acid by diluting 1.88 mL of glacial acetic acid in 23 mL of mQ water. Add 5 μl of 5000x ProteOrange stock solution and mix.

add mQ water add glacial acetic acidadd ProteOrange dye

Step 2.

Pour the prepared staining solution into a clean* pipet box or other suitable plastic dishes.

*Wash a pipet box well before use as detergents and other impurities can interfere with staining.

pour the staining solution

Step 3.

Place the gel into the staining solution.

Shake the gel for 30-60 minutes*. Cover the box with any lid or aluminum foil to protect the dye from light.

*for thinner gels or lower percentage gels, shorter times should be used; 60 min is optimal for 1 mm-thick 15% gel.

place the gel into the staining solutionplace the gel into the staining solutionshake the gel for 30-60 minutescover the box with any lid

Step 4.

Rinse the gel in a 7.5% acetic acid solution (containing no dye) for 30-60 seconds.

The gel is ready for visualization.

empty the pipet boxRinse the gel in a 7.5% acetic acid solutionRinse the gel for 30-60 seconds

Step 5.

For visualization, place the gel onto UV or blue-light transilluminator.

place the gel onto UV or blue-light transilluminatorview of the gel on the UV transilluminator

Use an appropriate optical filter and a highly sensitive camera to get the best result.

*This photo was taken using a 312 nm UV transilluminator and 575±25 nm optical filter.

proteins stained with ProteOrange

SYPRO™ is a trademark of Thermo Fisher Scientific

Coomassie™ is a trademark of Imperial Chemical Industries