Pierce™ Silver Stain Kit, 1 L - FAQs

以下是可能原因和解决方案：

- 染色的凝胶过度显色： 缩短显色时间。
- 忽略洗涤步骤或水质较差： 不要跳过和减少洗涤步骤，应使用超纯水洗涤。
- 所用设备被污染： 使用超纯水将设备冲洗干净。
- 制胶所用化学品不纯或预制胶已过期： 使用分析级化学品或使用未过期的预制胶。
- 终止液不能有效终止凝胶显色： 制备新的5%乙酸，并在凝胶孵育的最初几分钟里更换2次。

以下是可能原因和解决方案：

- 显色时间不足： 凝胶显色时间>5分钟，或加入新配制的显色工作液。
- 样品中的蛋白质含量极低或无蛋白质： 检测原始样品中的蛋白浓度。
- 溶液配制不恰当或遗漏了某些步骤： 检查溶液配制方法，并遵循操作步骤。
- 显色步骤前，水洗过度： 用超纯水洗涤凝胶3次，每次10分钟，然后重复染色步骤，并缩短该洗涤步骤。

可以。用水彻底清洗凝胶以除去乙酸，使用含有5%甘油和10%甲醇（或10%乙醇）的溶液浸泡凝胶1小时，然后在凝胶干燥装置内进行干燥。

该实验方案中的固定和染色时间非常灵活。凝胶固定至少30分钟后，可将其放在水中保存到第二天。凝胶染色时间控制在5分钟至24小时范围内，都不会使背景增强。

可以。首先，对Coomassie染色的凝胶进行脱色处理，以完全去除背景。如果使用了酸性或甲醇脱色溶液，则应使用超纯水彻底清洗凝胶，然后再使用Thermo Scientific银染试剂盒进行染色。

可以，点击这里（https://tools.thermofisher.com/content/sfs/brochures/TR0050-Stained-gels-for-MS.pdf）查看如何处理经过Thermo Scientific银染试剂盒染色的凝胶以用于质谱分析。然而，为获得最佳结果，我们推荐使用质谱分析用Thermo Scientific银染染料（货号24600）。胶内胰蛋白酶消化和质谱分析前进行凝胶染色和脱色所需的全部试剂和优化步骤在该试剂盒中均有提供。

该试剂盒可检测到蛋白质含量为0.4 ng的条带。大部分蛋白质在低纳克（1–5 ng）级含量时，均可轻松检测到。

该染料对大多数聚丙烯酰胺凝胶类型（即，不同供应商和不同缓冲液系统）的染色效果都很好。1D和2D聚丙烯酰胺凝胶均可使用该试剂盒染色。1.0mm凝胶的默认实验方案已经过优化；对于其他厚度的凝胶，需调整孵育和洗涤时间以获得最佳结果。

洗涤和固定凝胶，以除去电泳缓冲液中的盐。加入银染染料，释放银离子与蛋白质发生相互作用。加入显色剂，蛋白质条带随着结合银的减少而变黑。加入乙酸降低pH，终止显色。

Thermo Scientific银染试剂盒可检测蛋白质含量低至0.25ng的条带。

Thermo Scientific银染试剂盒可在室温下稳定保存1年。

Yes. References in the literature and molecular biology protocols indicate that nucleic acids can be detected in polyacrylamide gels with silver stains of this type.

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Yes. See Tech Tip: Process Stained Polyacrylamide Gels for Mass Spectrometry (https://assets.thermofisher.com/TFS-Assets/LSG/Application-Notes/TR0050-Stained-gels-for-MS.pdf). Alternatively, use our Thermo Scientific Silver Stain for MS (Cat No. 24600), which is the same silver stain kit but also includes destaining reagents and a protocol optimized for maximum protein recovery.

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SDS, glycine, amines and phosphates interfere with the staining method; this is why initial wash and fixing steps are necessary. Silver staining is also sensitive to pH conditions, chelators (sequester silver), and strong oxidants or reductants. To avoid these contaminants, use ultrapure water and clean equipment (avoid metal utensils), and avoid touching the gel except at the edges. Prepare working solutions of stain, sensitizer and developer immediately before use.

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The 1 minute sensitization and 2 x 1 minute water wash steps are important for optimum stain performance. Development time (2-3 minutes) is also critical, and stop solution must be added as soon as desired development occurs.

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The gel is washed and fixed to remove electrophoresis buffer salts. The silver stain is added, releasing silver ions that interact with the proteins. The developer is then added causing the protein bands to darken as the bound silver reduces. The color development is stopped by lowering the pH with acetic acid.

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Here are possible causes and solutions:

- Stained gel was overdeveloped. Reduce development time.
- Washing step(s) was missed or poor quality water was used. Do not skip or reduce wash steps and use ultrapure water.
- Contaminated equipment was used. Use clean equipment rinsed with ultrapure water.
- Impure chemical was used for gel preparation or precast gel has expired. Use analytical grade chemicals or use precast gels that have not expired.
- Stop solution was not effective in halting development of gel. Prepare new 5% acetic acid and replace it twice in the first minutes of incubation with the gel.

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Here are possible causes and solutions:

- Insufficient development time. Develop gel for more than 5 minutes or add freshly prepared Developer Working Solution.
- Minimal or no protein present in sample. Check protein concentration in the original sample.
- Improper solution preparation or skipped steps. Check solution preparation and follow procedure.
- Excessive water wash before development step. Wash gel 3 × 10 minutes with ultrapure water, then repeat staining procedure shortening this wash step.

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Yes. Thoroughly wash the gel with water to remove the acetic acid and then soak the gel in a solution containing 5% glycerol and 10% methanol (or 10% ethanol) for one hour before drying in a gel-drying apparatus.

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The protocol is quite flexible with regard to fixing and staining times. After gels are fixed for at least 30 minutes, they can be stored in water until the next day. The gel-staining time can range from 5 minutes to 24 hours without incurring additional background.

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Yes. First destain the Coomassie-stained gel to completely remove background. If an acid or methanol destaining solution was used, thoroughly wash the gel with ultrapure water, then proceed with the Thermo Scientific Silver Stain Kit protocol.

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Yes, see here (https://tools.thermofisher.com/content/sfs/brochures/TR0050-Stained-gels-for-MS.pdf) for the procedure for processing gels stained with Thermo Scientific Silver Stain, for mass spectrometry analysis. However, for best results, we recommend using the Thermo Scientific Silver Stain for Mass Spectrometry (Cat. No. 24600). This kit includes an optimized procedure and all the necessary reagents for staining gels and destaining gel pieces before in-gel trypsin digestion and mass spectrometry.

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A detection level of 0.4 ng per protein band has been achieved. Most proteins are easily detectable at low-nanogram (1-5 ng) amounts.

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The stain performs very well for most polyacrylamide gel types (i.e., suppliers and buffer systems). Both 1D and 2D polyacrylamide gels can be stained with this kit. The default protocol is optimized for gels that are 1.0mm thick; incubation and wash times may require adjustment to achieve optimal results for gels of other thicknesses.

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The gel is washed and fixed to remove electrophoresis buffer salts. The silver stain is added, releasing silver ions that interact with the proteins. The developer is then added causing the protein bands to darken as the bound silver reduces. The color development is stopped by lowering the pH with acetic acid.

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

The Thermo Scientific Silver Stain Kit detects proteins at 0.25 ng per band.

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We recommend storing the Thermo Scientific Silver Stain Kit at room temperature where it is stable for a year.

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