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查看更多产品信息 Colloidal Blue Staining Kit - FAQs (LC6025)
79 个常见问题解答
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。
与预染ladder中蛋白质结合的染料带有电荷并且与蛋白是共价结合的，因此，预染ladder的转印效率几乎总是高于SDS变性蛋白。所以，预染ladder不是衡量转印成功率最好的检测手段。此外，Invitrogen可逆性膜蛋白质染色试剂盒可对硝化纤维素和PVDF膜上的蛋白质进行完全且可逆地[的]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），并可在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，并且在某些情况下可达到更高的灵敏度。
可以，胶体蓝染料可在免疫印迹前使用。但是，为得到理想的转印效率，我们建议进行凝胶脱色处理，然后在一系列Tris 碱/甘氨酸/SDS溶液中平衡，增加染料的溶解；转印完成时，应使用甲醇处理膜，从而在之前去除染料[从而在化学发光显影前去除染料]（不需要在化学发光检测前进行脱色处理）。
丙烯酰胺浓度低于10%的凝胶，通常背景较高，因为低比例丙烯酰胺凝胶具有较大的孔，胶体可渗入和停留在这些孔中。去除多余背景的方法为，将凝胶放在25%甲醇溶液中孵育，直至获得干净的背景。应注意，这种方法也会去除条带上的部分染色剂。在浓度>25%的甲醇溶液中长时间孵育，可导致蛋白质条带和背景完全脱色。
可以，您可以用水将凝胶完全脱色后从头开始染色操作，或者，如果您感觉染色力度还是有点低并且想要加深染色，可将凝胶直接放回染色液中。
胶体，或蓝色块状物，在这种染色操作中出现是正常的。但是，如果含量异常高，通常是因为甲醇加入量太少或未加入甲醇。
可能不会。但是，如果Stainer B溶液的加入量比要求加入的量少10%或更多，可能导致染色力度不足。加入过多的Stainer B溶液不会影响结果，Stainer A溶液增加/减少50%也不会影响结果。
当胶体蓝Stainer A与Stainer B溶液混合时，可能会形成沉淀；轻轻摇晃30秒，沉淀即可溶解。
不，您看到的蓝色块状物被称为胶体。它们使染色剂能够有效染色。我们建议在使用前摇晃溶液，使瓶中的块状物均匀散开。
低比例丙烯酰胺凝胶的背景较高，是因为这些凝胶具有较大的孔，胶体可渗入和停留在孔中。
去除背景的方法为，将凝胶放在25%甲醇溶液中孵育，直至获得干净的背景。应注意，这种方法也会去除一部分染色剂。
在浓度>25%的甲醇溶液中长时间孵育，可使蛋白质条带和背景完全脱色。
如果采用NuPAGE Bis-Tris/Small Peptides实验方案，可能会使背景降低。该方案利用额外的固定步骤去除多余SDS；SDS可作为抗胶体试剂，使背景升高。染色液的低pH条件可固定凝胶，但固定速度没有NuPAGE方案中的预固定步骤快。
可以，胶体蓝染色试剂盒对Invitrogen Zymogram凝胶染色的效果很好。
Coomassie G250比Coomassie R250更灵敏，而Coomassie R250比Coomassie G250的染色速度更快。
胶体蓝染料中的某些成分可能过于粗糙，不宜用于蛋白质测序，但Coomassie染料本身并不会产生不利影响。
胶体蓝Coomassie G250染料需要610 nm波长，而Coomassie R250染料需要588 nm波长。
不能，加热会使胶体溶解，进而使溶液中的游离染料增加，导致背景升高和染色强度降低。
不可以，染色溶液应在使用前24小时以内配制。如果未使用新鲜的溶液，会产生较高的染色背景。
需要，固定有两个目的：固定凝胶中的样品和帮助去除凝胶背景。如果不使用固定液，凝胶背景会很高，检测灵敏度降低。凝胶中的两性电解质残留会导致高背景。
胶体蓝染色试剂盒可以保存在冰箱中，但在使用前应恢复到室温。同时，Stainer B试剂在使用前应摇匀。冷藏不会延长染料的保质期。
5%漂白剂能有效清除塑料、瓷器和金属表面的胶体蓝染料。
请看下述建议：
- 染色凝胶在预处理凝胶干燥溶液（如Gel-Dry溶液）中的时间不能超过5分钟。
- 暴露于预处理凝胶干燥溶液的时间过长，会导致凝胶完全脱色。
不能，胶体蓝染色试剂盒的化学成分只能用于蛋白质和多肽染色。SilverXpress染色试剂盒是高度灵敏的核酸染料。
胶体蓝染色试剂盒使用Coomassie G-250，而标准Coomassie染色方案使用Coomassie R-250。Coomassie G-250为蓝绿色，而Coomassie R-250接近于深蓝色。两种染料的区别在于染料本身的色调，而非染色强度。
有区别，该产品对非还原型蛋白质的染色更强。
不能，我们不建议使用胶体蓝（G-250）染色试剂盒进行膜染色，因为这会产生非常高的背景。更好的方案有：
- 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。
- SimplyBlue SafeStain。SimplyBlue SafeStain使用手册中有关于PVDF染色的实验方案，但不推荐将其用于硝化纤维素膜，因为会产生高背景。
- 酰胺黑：与Coomassie相同，但灵敏度更低。
- 丽春红S：与Coomassie相同，但灵敏度更低。
- 紫外透照法：转印后，将膜放在滤纸上，使其在室温下干燥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。
- Invitrogen可逆性膜蛋白质染料（货号IB7710）：可对硝化纤维素和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。
经胶体蓝染料染色的蛋白质，可用于质谱（MS）分析。
胶体蓝染色试剂盒的灵敏度比传统Coomassie Blue染色技术强5倍。使用胶体蓝染色试剂盒，可在1小时内检测到4–20% 1.0 mm Invitrogen Tris-甘氨酸凝胶上<10 ng的BSA。非还原性样品的染色强度略高于还原性样品。在染色溶液中浸泡1小时后，可看见条带。
胶体蓝染色试剂盒推荐室温保存，可在室温下稳定保存1年。
丙烯酰胺比例低于10%的凝胶具有较大的孔，胶体会渗透入孔并将其封闭，从而产生较高的背景。为降低过高的背景，可将凝胶在25%甲醇溶液中孵育，直至获得清晰的背景。应注意，这样做会部分去除条带上的染料，在>25%甲醇溶液中长时间孵育会导致蛋白质条带和背景完全脱色。
使用NuPAGE Invitrogen Bis-Tris凝胶/小肽附带的实验方案，可能会降低背景。该实验方案包含额外的固定步骤以除去多余的SDS，SDS是一种抗胶体试剂，可导致较高背景。染色液的低pH条件可固定凝胶，但没有NuPAGE实验方案特有的预固定步骤速度快。
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.
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.
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.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
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.
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.
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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.
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.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
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.
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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.
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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.
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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.
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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.
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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.
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Yes, it stains non-reduced proteins more intensely.
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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:
- 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.
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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.
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We recommend storing the Colloidal Blue Staining kit at room temperature where it is stable for 1 year.
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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.
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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.
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