SimplyBlue™ SafeStain

Coomassie G-250 will give a sharp dye front with both NuPAGE MES and MOPS Running Buffers and is therefore used as the tracking dye in the NuPAGE LDS Sample Buffer.

Bromophenol blue runs more slowly than some peptides with the NuPAGE MES Running Buffer system.

Coomassie G-250 migrates much closer to the moving ion front than bromophenol blue, ensuring that small peptides will not be run too far (e.g., off the gel).

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

The Colloidal Blue Staining Kit (Cat. No. LC6025) is best for quantitation by densitometry. You can also use SimplyBlue SafeStain for this application.

The great advantage of SimplyBlue SafeStain is that it is very easy to use and safe.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

Check the cap on the bottle. If the bottles are not tightly sealed, the alcohol can evaporate from the stain causing substantial decrease in stain sensitivity.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

After staining with SimplyBlue SafeStain, use deionized water for the less strongly retained protein bands on the PVDF membrane.

Increasing methanol or ethanol concentrations up to 70% should destain any remaining bands. You can leave the membrane in the destain indefinitely.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

Membranes cannot be stained with Colloidal Blue, the background will be too high. Better alternatives include:

1) Coomassie (non-colloidal) staining: Stain in 0.1% Coomassie Blue R-250 in 50% methanol for 5 min and destain with several changes of 50% methanol and 10% acetic acid. Rinse with several changes of water, air dry and store for up to 12 months at -20°C. Sensitivity is approximately at the 50-100 ng level.

2) SimplyBlue SafeStain. There is a protocol included in the SimplyBlue manual for staining PVDF but it is not recommended for nitrocellulose because of the high background.

3) Amido Black: less sensitive than Coomassie Blue.
Recipe for amido black: (1L) 450 mL methanol, 450 mL dH2O, 100 mL glacial acetic acid, 0.1 g amido black.
Procedure: combine ingredients and stir until the amido black is dissolved. If the membrane has dried up, pre-wet by floating on dH2O and soaking for 5 min. Transfer to tray containing amido black for 5-10 min. Wash in several changes of dH2O.

4) Ponceau S: less sensitive than Coomassie Blue.
Recipe for Ponceau S (10X stock): 2 g Ponceau S, 30 g trichloroacetic acid, 30 g sulfosalicylic Acid, dH2O to 100 mL
Combine ingredients and stir until Ponceau S is dissolved. Dilute 1:10 before using.
Procedure: If membrane has dried up, pre-wet membrane by floating on dH2O and soaking for 5 min. Transfer membrane to tray containing Ponceau S and incubate for 5-10 min. Wash membrane in several changes of dH2O.

5) UV transillumination: Place membrane on filter paper after blot is finished and allow to dry at room temperature for about 10 min. Rewet in 20% methanol and view the blot in front of white light while it is still wet; the bands will look more translucent than the membrane. If the bands disappear because they dry, rewet again.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.