Colloidal Blue Staining Kit - FAQs

It is not recommended because the background will be too high. Better alternatives include: 1) Invitrogen Reversible Membrane Protein Stain Kit (Cat. No. IB7710).

2) 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.

3) Use SimplyBlue SafeStain (Cat. No. LC6060). The SimplyBlue SafeStain manual has the protocol for staining PVDF membranes, but it is not recommended for nitrocellulose because of high background.

4) Amido Black: same as Coomassie but less sensitive.

5) Ponceau S: same as Coomassie but less sensitive.

6) 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 as the membranes dries, rewet again.

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

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.

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

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.

It may be possible to reduce background by using the protocol given for NuPAGE Invitrogen Bis-Tris/Small Peptides.

This protocol incorporates an extra fix step to remove excess SDS, which can act as an anti-colloidal agent and lead to higher background.

The low pH of the staining solution will fix the gel, but not as fast as the pre-fix step specified in the NuPAGE protocol.

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.

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

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

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

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.

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

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.

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.

The dye that is bound to the proteins in the pre-stained ladder is charged and covalently bound, so the transfer efficiency of pre-stained ladders is almost always better than that of SDS-denatured proteins. Therefore, pre-stained ladders are not a good measure of transfer success. On the other hand, the Invitrogen Reversible Membrane Protein Stain allows for complete, reversible staining of protein on nitrocellulose & PVDF membranes. The staining 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.

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

Background is generally higher in gels less than 10% in acrylamide concentration due to penetration and trapping of colloids within the large pores of the low-percentage acrylamide gels. Excess background may be removed by incubating in 25% methanol solution until a clear background is obtained. Be aware that dye will also be partially removed from the bands and prolonged incubation in >25% methanol will result in a complete destaining of protein bands and background.

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

Yes, you can either completely destain the gel in water and start staining from scratch or if you feel that the staining intensity is a little low and you would like to darken it, you can directly place the gel back in the staining solution.

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

Colloids, or blue chunks, are routine in this type of staining procedure. However, if the amount is abnormally high, it usually indicates that too little or no methanol was added.

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Probably not, however if you add greater than 10% less than the total required amount of Stainer B solution, your gel may not stain as intensely as it would have with the proper amount. Adding more of the Stainer B solution will not affect results, nor will +/- 50% of the Stainer A solution.

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When Colloidal Blue Stainer A and Stainer B solutions are combined, a precipitate may form; this will dissolve within 30 seconds of gentle shaking.

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No, the blue “chunks” you see are called colloids. They enable the stain to work effectively. We recommend shaking the solution well before using to evenly distribute these chunks throughout the bottle.

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

- The background is higher in low percentage acrylamide gels due to penetration and trapping of colloids within the large pores of these gels.
- Background may be removed by incubating the gel in 25% methanol solution until a clear background is obtained. Be aware that dye will also be partially removed from the bands.
- Prolonged incubation in >25% methanol results in complete destaining of protein bands and background.

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

A reduction in background may be seen if the protocol for NuPAGE Bis-Tris/Small Peptides is used instead. It uses an extra fixing step to remove excess SDS, which can act as an anti-colloidal agent and cause higher background. The low pH of the staining solution will fix the gel, but not as fast as the pre-fixing step performed in the NuPAGE protocol.

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

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

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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Some of the components of the Colloidal Blue Stain are probably too harsh to be used in conjunction with protein sequencing, although the actual Coomassie dye itself does not have an adverse effect.

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

Colloidal Blue Coomassie G250 Stain requires a wavelength of 610 nm whereas standard Coomassie R250 stains require a wavelength of 588 nm.

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No, heat causes colloids to solubilize; this in turn, causes the free-dye in solution to increase which causes an increase in background and a decrease in staining intensity.

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No, the staining solution should be prepared fresh, no more than 24 hours before use. If fresh solution is not used, staining background will be high.

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Yes, the fix serves two purposes: it fixes the sample in the 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 can be caused by ampholytes remaining in the gel.

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

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.

A 5% bleach solution will effectively remove the Colloidal Blue stain from plastic, porcelain and metal surfaces.

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Please see the following recommendations:

- Do not leave the stained gel in pretreatment gel drying solutions, such as Gel-Dry Solution, for more than 5 minutes.
- Prolonged exposure to pre-treatment gel drying solutions will destain the gel completely.

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 stains only proteins and peptides. The SilverXpress Staining Kit is a highly sensitive stain for nucleic acids.

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

The Colloidal Blue Staining Kit uses Coomassie G-250 whereas standard Coomassie staining protocols use Coomassie R-250. Coomassie G-250 is a turquoise color whereas Coomassie R-250 is closer to navy blue. The difference is in the shade of color, rather than the intensity.

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

Yes, it stains non-reduced proteins more intensely.

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:

- 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 PVDF but it is not recommended for nitrocellulose because of the high background.
- Amido Black: same as Coomassie but less sensitive.
- Ponceau S: same as Coomassie but less sensitive.
- UV transillumination: After blotting, place the membrane on a filter paper and allow to 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.
- Invitrogen Reversible Membrane Protein Stain (Cat# IB7710): Allows for complete, reversible staining of protein on nitrocellulose & PVDF membranes. Sensitivity is higher than Ponceau S (less than 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.

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

Proteins stained using Colloidal Blue Stain are compatible with mass spectrometry (MS) analysis.

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The Colloidal Blue Stain is five times more sensitive than traditional Coomassie Blue staining techniques. Using the Colloidal Blue Staining Kit, less than 10 ng of BSA can be detected on a 4-20% 1.0 mm Invitrogen Tris-Glycine Gel in 1 hour. Non-reduced samples stain slightly more intensely than reduced samples. Bands are visible after 1 hour in staining solution.

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

We recommend storing the Colloidal Blue Staining kit at room temperature where it is stable for 1 year.

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

Background is generally higher in gels with less than 10% acrylamide percentage due to penetration and trapping of colloids within the large pores of these gels. Excess background may be reduced by incubating the gel in 25% methanol solution until a clear background is obtained. Be aware that the dye will also be partially removed from the bands and that prolonged incubation in >25% methanol will result in complete destaining of protein bands and background.

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

It may be possible to reduce background by using the protocol provided for NuPAGE Invitrogen Bis-Tris gels/Small Peptides. This protocol incorporates an extra fix step to remove excess SDS, which can act as an anti-colloidal agent and lead to higher background. The low pH of the staining solution will fix the gel, but not as fast as the pre-fix step specified in the NuPAGE protocol.

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