Pierce™ Coomassie Brilliant Blue R-250 Dye, 50 g - FAQs

When dissolved in 0.01 M citrate buffer at pH 3.0, it has an absorption maximum at 555 nm; the protein-dye complex is characterized by a peak slightly broader than that of the free dye with an absorption maximum at 549 nm.

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In acidic conditions, Coomassie dye primarily binds basic amino acids (arginine, lysine and histidine).

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Add 100 mL of glacial acetic acid to 450 mL distilled water.
Dissolve 3 g of Coomassie R-250 dye in 450 mL methanol.
Mix the acetic acid and methanol solutions.
Filter the solution before use.

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Yes, Imperial Protein Stain (Cat. Nos. 24615 (1L) and 24617 (3 x 1L) is a ready-to-use colorimetric stain formulated with Coomassie R-250 dye that delivers consistent nanogram-level detection of proteins in polyacrylamide electrophoresis gels.

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Coomassie G-250 has an additional methyl group on the lower two central rings in the structure, as compared to Coomassie R-250. Bands stained with Coomassie R-250 are reddish-purple in color; Coomassie G250 bands are greenish-blue in color.

Sometimes referred to as colloidal Coomassie Blue, the G-250 form differs from R-250 in more readily forming dye-dye aggregates in solution. Consequently, G-250 interacts more weakly with polyacrylamide gel matrix than with itself, resulting in lower background that can be more easily washed away.

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No we do not recommend staining membranes with the Colloidal Blue (G-250) Staining Kit as the background will be too high. Better alternatives include:

- Invitrogen Reversible Membrane Protein Stain (Cat. No. IB7710): Allows for complete, reversible staining of protein on nitrocellulose & PVDF membranes. Sensitivity is higher than Ponceau S (<10 ng of BSA in 10 mins as blue bands) and the staining is reversible in 5 minutes. Western blot detection is unaffected by the staining and erasing process, and in some cases higher sensitivity is achieved.
- Coomassie (non-colloidal) staining: Stain in 0.1% Coomassie Blue R-250 in 50% methanol for 5 minutes 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 degrees C. Sensitivity is approximately at the 50-100 ng level.
- SimplyBlue SafeStain. There is a protocol included in the SimplyBlue SafeStain manual for staining dry PVDF membranes but it is not recommended for wet PVDF membranes or nitrocellulose because of the high background.
- Amido black: same as Coomassie stain but less sensitive. Please see protocol in the manual (https://tools.thermofisher.com/content/sfs/manuals/invitrolonpvdf_man.pdf).
- Ponceau S: same as Coomassie stain but less sensitive. Please see protocol in the manual (https://tools.thermofisher.com/content/sfs/manuals/invitrolonpvdf_man.pdf).
- UV transillumination: After blotting, place the membrane on a filter paper and allow to air-dry at room temperature for about 10 minutes. 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 the membrane is dry, rewet the membrane.

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