SimplyBlue™ SafeStain, 3.5 L - FAQs

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.

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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.

Coomassie sensitivity: 50-500 ng protein per band
Silver-staining sensitivity: 1-5 ng protein per band
In general, silver staining is 10-100 times more sensitive than Coomassie staining.

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The Coomassie dye binds to the protein via hydrophobic interactions, so anything that can disrupt these interactions can potentially serve to remove the dye.

For mild removal conditions, try nonionic detergents such as Triton X-100. If harsher conditions are acceptable, organic solvents like acetonitrile or alcohols should be effective.

Many times, washing with 30% ethanol will remove the stain. Changing the pH or salt concentration is not expected to help.

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

Yes, the fix serves two purposes: it fixes the sample in the IEF gel and it helps to remove gel background.

If you do not use the fixing solution, the background on the gels will be high and detection will be less sensitive.

High background is caused by ampholytes remaining in the gel.

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

Yes, either Coomassie or Colloidal Blue stain can be used before Western blotting.

However, for optimal transfer efficiency, destain the gel and then equilibrate in a series of Tris base/Glycine/SDS solutions to increase solubility;

When the transfer is complete, the membrane is treated with methanol to remove the stain prior to chromogenic development (not necessary prior to chemilumninescent detection). Review Current Protocols in Molecular Biology Unit 10.8.

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The precipitate is caused by a temporary high concentration of one of the components in a localized area.

Gentle mixing of the solution allows the precipitate to go into solution.

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The blue particles are called colloids. They are what enables the kit to work effectively.

Just remember to shake the solution well before using to evenly distribute them throughout the bottle.

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The Colloidal Blue Staining kit can be stored in the refrigerator, but be sure to let the kit return to room temperature before use.

Also, remember to shake the Stainer B reagent prior to use. Cold storage will not enhance the shelf-life of the stain.

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

No, due to its chemistry, the Colloidal Blue staining kit only stains proteins and peptides.

The Colloidal Blue Staining Kit uses G-250 Coomassie whereas standard Coomassie staining protocols use R-250.

The G-250 is a turquoise color whereas R-250 is closer to navy blue. The difference is in the shade of color, rather than the intensity.

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Yes, the Colloidal Blue Staining kit works very well with Invitrogen Zymogram gels.

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These gels are for basic proteins, where they will migrate to the cathode. The interfering substances are urea and Triton X-100.

To improve results, the gels can be rinsed three times (5 min each) with water after the methanol/acetic acid fix step, before adding the staining solution.

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The G-250 coomassie dye is very soluble in alcohol. To remove all of the stain, incubate the gel in a 10-30% ethanol solution for approximately 15 min or until clear. Rinse and rehydrate the gel in pure water and proceed from there.

In the Colloidal Blue Staining kit manual, it is recommended to incubate the stained gel in GelDry (a 30% alcohol solution) for only 5 min as opposed to the normal 15 min because a 15 min incubation would completely "bleach" the gel.

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

SimplyBlue SafeStain is completely non-hazardous and does not require methanol or acetic acid fixatives or destains. Risk of hazardous exposure and unpleasant odors are eliminated.

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

If you need to leave the gel overnight in the stain, add 2 mL of 20% NaCl (w/v) in water for every 20 mL of stain. This procedure will not affect sensitivity.

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

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.

Yes, Colloidal Blue Stain can be used before western blotting. However, for optimal transfer efficiency, we recommend destaining the gel and then equilibrating in a series of Tris base/Glycine/SDS solutions to increase solubility; when the transfer is complete, the membrane should be treated with methanol to remove the stain prior to chromogenic development (not necessary prior to chemiluminescent detection).

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

The problem is related to the TCA not being rinsed off as it lowers the pH of the solution and causes aggregation of the stain. We recommend washing in large volumes of water, twice for 5 minutes each and performing the last wash for at least an hour (can even go overnight if needed). Another recommendation is to use excess amount of SimplyBlue SafeStain.

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

Coomassie G250 is more sensitive than Coomassie R250 whereas Coomassie R250 stains faster than Coomassie G250.

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You can use SimplyBlue SafeStain to stain the PVDF membrane to detect your protein to be sequenced. After detection and prior to sequencing, rinse the membrane in 20-30% ethanol to remove the remaining stain.

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We recommend leaving the gel in the stain overnight. There is no need to add the extra salt solution as recommended in the manual because there are no buffer salts and SDS to worry about, and that would end up inhibiting the staining too much.

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

There should be no problem with using a SimplyBlue stained protein band for immunization. The process of staining and destaining the gel should remove SDS (if an SDS PAGE gel is used) so it will not interact with the Freund's Adjuvant.

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Our recommendation would be to separate the protein from the Coomassie G250 stain by electroelution or alcohol treatment so as to not illicit an immune response to the Coomassie G250. If the antibodies are to be used in membrane-based immunodetection, then inserting the membrane-bound band subcutaneously can produce antibodies that work especially well in membrane-based detection.

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SimplyBlue Safestain is specially designed for safe, non-hazardous disposal. It does not contain methanol or acetic acid and does not require the use of methanol or acetic acid which must be disposed of as hazardous waste.

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SimplyBlue SafeStain does not contain acetic acid or methanol. An alcohol/acetic acid fixing step prior to staining with SimplyBlue SafeStain is not required or recommended.

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Proteins stained using the SimplyBlue SafeStain are compatible with mass spectrometry (MS) analysis.

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SimplyBlue SafeStain can be used to stain dry PVDF membranes. Staining nitrocellulose and wet PVDF membranes results in high background and is not recommended.

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With the regular staining protocol, 7 ng of BSA can be detected after destaining whereas with the microwave staining protocol, 5 ng of BSA can be detected.

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SimplyBlue SafeStain is a ready-to-use proprietary staining solution that contains Coomassie G50. It does not contain acetic acid or methanol.

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We recommend storing the SimplyBlue SafeStain at room temperature where it is stable for one year from date of shipment.

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

You can use the SimplyBlue SafeStain for protein quantitation by densitometry. However, the Colloidal Blue Staining Kit (Cat. No. LC6025) is the best suited for this application.

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