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Directions for Use Silver Staining – Silver De-Staining Directions for Use Silver Staining – Silver De-staining Kit Code Description Size M335-KIT Silver Staining – Silver De-Staining Kit 12 mini-gels General Information The Silver Staining – De-staining Kit is a combination of two products for convenience; Silver BULLit™, Silver Stain Kit (for 50 mini-gels) and Silver Subtract™, Silver De-staining Reagent (for 12 mini-gels). Silver BULLit™, Silver Stain Kit can be used to detect proteins in polyacrylamide gels with high sensitivity and nearly undetectable background. The kit's colorimetric staining procedure allows detection of sub-nanogram levels of protein, which is 100- fold more sensitive than Coomassie® blue staining. The staining procedure begins with a fixation step to reduce band diffusion and remove interfering substances and is followed by a sensitization process, which enhances binding of silver ions to proteins. The staining step allows silver ions to permeate the gel and bind preferentially to sulfhydryl and carboxyl side chains. In the final development step, silver ions are reduced to metallic silver to form precipitates that are visible as brown bands within the gel. Silver Subtract™ Silver De-staining Reagent completely de-stains gels stained with VWR Life Science AMRESCO’s Silver BULLit™, Silver Stain Kit or any other silver staining product. It is an excellent kit for the removal of silver ions before re-staining with silver stain. Silver Subtract™ is also compatible with subsequent visualization of the gel with fluorescent or chemiluminescent stains, or with chromogenic stains such as Coomassie® blue. Partial de- staining can also be achieved with more dilute reagents to remove high background, reduce staining of overloaded gels, or remove artifacts and uneven background. Gels re-stained after using Silver Subtract™ do not exhibit a decrease in band intensity or increase in background. Silver BULLit™, Silver Stain Kit offers ultra-sensitive protein detection with clear background Silver Subtract™, Silver-De-staining Reagent eliminates the need to rerun over stained, blotched or fingerprinted gels and works in as little as 5 minutes AMRESCO, LLC Corporate Headquarters, 28600 Fountain Parkway, Solon, OH 44139 Directions for Use Storage/Stability Store at room temperature (18 – 26°C). Stable 1 year when stored as recommended in the original packaging. Product Use Limitations For research use only. Not for therapeutic or diagnostic use. Materials Supplied Silver BULLit™, Silver Stain Kit K490-250ML Silver Stain, 10X Concentrate K491-250ML Sensitizer, 10X Concentrate K492-250ML Developer, 5X Concentrate (x2) Silver Subtract™, Silver De-staining Reagent M321-25ML Silver Subtract™ Solution A, 25X M320-25ML Silver Subtract™ Solution B, 25X Required Materials Not Supplied Methanol Ethanol 37% formaldehyde Glacial acetic acid Distilled / deionized water Glass staining tray Oribtal shaker AMRESCO, LLC Corporate Headquarters, 28600 Fountain Parkway, Solon, OH 44139 Directions for Use Protocol/Procedure Silver BULLit™, Silver Stain Kit Notes: Staining intensity varies by the type of protein in the sample. Acidic proteins and proteins with highly negatively charged sulfated sugar residues, such as proteoglycans and mucins, are not readily detected by silver stains. Perform all steps at room temperature using freshly prepare solutions. Use distilled / deionized water for reagent preparation. Wear powder-free gloves for all steps to avoid fingerprints or other artifacts on the gel. Rinse gloves with distilled, deionized water between steps. Reagent Preparation Fixative (100 mL) 35% Ethanol (300 mL) Component Volume (mL) Component Volume (mL) Methanol 50.00 95% ethanol 81.00 Glacial acetic acid 12.00 Distilled / Deionized Water 219.00 37% formaldehyde 1.35 Distilled / Deionized Water 36.65 1X Sensitizer Solution (100 mL) 1X Stain Solution (100 mL) Component Volume (mL) Component Volume (mL) Sensitizer,10X Concentrate 10.00 Silver Stain, 10X Concentrate 10.00 Distilled / Deionized Water 90.00 Distilled / Deionized Water 90.00 1X Developer Solution (100 mL) Stop Solution (100 mL) Component Volume (mL) Component Volume (mL) Developer, 5X Concentrate 20.00 Methanol 50.00 Distilled / Deionized Water 80.00 Glacial acetic acid 12.00 Distilled / Deionized Water 38.00 AMRESCO, LLC Corporate Headquarters, 28600 Fountain Parkway, Solon, OH 44139 Directions for Use Silver staining of protein mini-gel 1. After electrophoresis, wash the gel 2 x 5 minutes in distilled/deionized water. 2. Place the gel in a clean glass staining tray with 100 mL Fixative solution. Incubate for 2 hours to overnight with gentle agitation. 3. Remove the Fixative solution and wash the gel three times (3X) for 20 minutes each in 100 mL of 35% Ethanol with gentle agitation. 4. Remove the Ethanol wash and add 100 mL of 1X Sensitizer solution. Incubate for 2 minutes with gentle agitation. 5. Remove the 1X Sensitizer solution and wash the gel three times (3X) for 5 minutes each in distilled/deionized water with gentle agitation. 6. Remove the water wash and add 100 mL of 1X Stain Solution. Incubate the gel for 20 minutes with gentle agitation. 7. Remove the 1X Stain Solution and wash the gel two times (2X) for 1 minute each in distilled/deionized water with gentle agitation. 8. Remove the water wash and add 100 mL of 1X Developer Solution and incubate with gentle agitation until the bands become visible (approximately 10 minutes). Bands should appear dark brown against a pale background. Note: The rate of band development is temperature-dependent. If the gel is over-developed, artifacts are present, the background is too dark, or if the bands are over-stained, the gel may be destained with VWR Life Science AMRESCO’s Silver Subtract™, Silver De-staining Reagent and restained as desired. 9. Remove the 1X Developer Solution and add 100 mL of Stop Solution. Incubate the gel for 20 minutes with gentle agitation to prevent further color development. 10. The gel may be stored at 4ºC in 1% Acetic Acid. AMRESCO, LLC Corporate Headquarters, 28600 Fountain Parkway, Solon, OH 44139 Directions for Use Silver Subtract™, Silver De-staining Reagent Notes: Silver-stained gels stored in 1% acetic acid should be thoroughly rinsed in deionized/distilled water before destaining. The de-staining solution should be prepared immediately before use. Complete destaining of silver-stained gels 11. Prepare 50 mL of 1X de-staining solution per mini-gel. Component Volume Silver Subtract™ Solution A, 25X 2 mL Silver Subtract™ Solution B, 25X 2 mL Deionized/Distilled Water 46 mL 12. Completely submerge the gel in the 1X de-staining solution and gently agitate until the desired level of destaining is achieved. Note: Silver-stained bands are typically destained completely within 5 minutes. 13. Stop the de-staining reaction by washing the gel copiously with deionized/distilled water. Partial de-staining of silver-stained gels Notes: Slower, more controlled de-staining can be achieved by decreasing the final concentration of the staining reagents to 0.1X or 0.05X. These more diluted de-staining solutions may be used to retain silver-stained bands while reducing background staining and artifacts. 1. Prepare 50 mL of 0.1X or 0.05X de-staining solution per mini-gel. 0.1X Solution Component Volume Silver Subtract™ Solution A, 25X 0.2 mL Silver Subtract™ Solution B, 25X 0.2 mL Deionized/Distilled Water 49.6 mL 0.05X Solution Component Volume Silver Subtract™ Solution A, 25X 0.1 mL Silver Subtract™ Solution B, 25X 0.1 mL Deionized/Distilled Water 49.8 mL AMRESCO, LLC Corporate Headquarters, 28600 Fountain Parkway, Solon, OH 44139 Directions for Use 2. Completely submerge the gel in the 0.1X or 0.05X de-staining solution and gently agitate until the desired level of de-staining is achieved. 3. Stop the de-staining reaction by washing the gel copiously with deionized/distilled water. Frequently Asked Questions Can the Developer, 5X Concentrate be used if it contains crystals? Crystals may form in the Developer, 5X Concentrate during storage. Before use, warm the solution gently until the crystals are re-dissolved. Can the Silver Stain, 10X Concentrate be used if it has a cloudy appearance? The Silver Stain, 10X Concentrate may appear cloudy or contain a fine precipitate, but this appearance will not affect the performance of the stain. Why is the background staining dark? There may be residual acid in the gel. o Increase the washing time after the fixation step. The gel was made using poor quality acrylamide. o Acrylamid quality can affect the background appearance of a silver-stained gel. Use high quality acrylamide, such as VWR Life Science AMRESCO’s Acryl/Bis™ 37.5:1, 40% Solution (part number 0254). Poor quality water was used in all the solutions in the protocol. o Use fresh, distilled/deionized water in all solutions. The gel was not washed sufficiently. o Follow the recommended washing procedure without shortening or skipping wash steps. Ensure the volumes used for washing are sufficient to submerge the gel and allow free movement during gentle agitation. Dirty equipment was used. o Use only clean equipment rinsed with high quality water. Can plastic trays be used for staining instead of glass or stainless steel? Plastic trays are not recommended for silver staining as they may contain compounds that reduce silver nitrate to metallic silver. Why are there streaks or yellow background on the gel? AMRESCO, LLC Corporate Headquarters, 28600 Fountain Parkway, Solon, OH 44139 Directions for Use Streaks or yellow background may appear due to an excess of reducing agents, such as 2-mercaptoethanol or DTT. o Reduce the amount of reducing agent in the sample buffer. Why are the bands faint or undetected? Development time may be insufficient or the working Developer solution may not be fresh. o Always use fresh 1X Developer solution. Develop the gel for > 5 minutes. There may be insufficient protein in the sample. o Confirm the protein concentration of the protein sample before use.
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