Pierce™ Silver Stain Kit, 1 L - FAQs

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.

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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.

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