Q: What is the absorption maximum of free and protein-bound dye Coomassie R-250?

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

Q: To what amino acids does Coomassie R-250 bind?

In acidic conditions, Coomassie dye primarily binds basic amino acids (arginine, lysine and histidine). Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center .

Q: How do I prepare a solution of Coomassie R-250 dye?

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

Q: Is Coomassie Brillant Blue R-250 available already prepared in solution?

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

Q: What are the differences between R-250 and G-250 Coomassie Dyes?

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

Question 1

转印后的膜能否用胶体蓝（G-250）染色试剂盒进行染色？

Accepted Answer

不能。我们不建议使用胶体蓝（G-250）染色试剂盒对膜进行染色，因为这会产生很高的背景。更好的染色方法包括：

1.Invitrogen可逆性膜蛋白质染料（货号IB7710）：可对硝化纤维素膜和PVDF膜上的蛋白质进行完整的可逆性染色。该产品的灵敏度高于丽春红S（在10分钟内检测到蓝色条带上<10 ng的BSA），可在5分钟内实现可逆性染色。免疫印迹检测不受染色和清除染料过程的影响，在某些情况下可达到更高的灵敏度。
2.Coomassie（非胶体）染色：使用溶于50%甲醇的0.1% Coomassie Blue R-250染色5分钟，然后用50%甲醇和10%乙酸洗涤多次进行脱色。用水洗涤多次、风干并在-20℃下保存，最长可保存12个月。灵敏度约为50-100 ng。
3.SimplyBlue SafeStain染液。SimplyBlue SafeStain使用手册中有对干燥PVDF膜进行染色的实验方案，但不推荐将其用于湿润的PVDF膜或硝化纤维素膜，因为会产生较高的背景。
4.酰胺黑染液：与Coomassie相同，但灵敏度更低。请参见使用手册中的实验方案。
5.丽春红S染液：与Coomassie相同，但灵敏度更低。请参见使用手册中的实验方案。
6.紫外透照法：转印后，将膜放在滤纸上，在室温下干燥10分钟。在20%甲醇中润湿，在湿润状态下于白光前观察印迹；条带会比膜看起来更透明。如果条带随膜变干而消失，则再次润湿膜。

Question 2

What is the absorption maximum of free and protein-bound dye Coomassie R-250?

Accepted Answer

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.

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

Question 3

To what amino acids does Coomassie R-250 bind?

Accepted Answer

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

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

Question 4

How do I prepare a solution of Coomassie R-250 dye?

Accepted Answer

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.

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

Question 5

Is Coomassie Brillant Blue R-250 available already prepared in solution?

Accepted Answer

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.

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

Question 6

What are the differences between R-250 and G-250 Coomassie Dyes?

Accepted Answer

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.

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

Question 7

Can membranes be stained with the Colloidal Blue (G-250) Staining Kit after blotting?

Accepted Answer

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.

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