SilverQuest™ Silver Staining Kit - FAQs

In general, the required amount needed to identify a gel-separated protein by enzymatic digestion (usually Trypsin) is about 2 pmole (equivalent to approximately 0.12 µg). Our SilverQuest Silver staining kit is mass spectrometry compatible.

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The sensitizing step is the optimal point to stop the procedure. The gel can be left overnight in the Sensitizing Solution. Although some other kits recommend leaving gels in the fix step, we have found that overnight fixation diminishes stain performances.

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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.

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This is usually due to overloading of the protein sample. We recommend decreasing the protein load per band. For an unknown protein, a serial dilution may be necessary to determine the best amount to load for a particular protein.

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Here are possible causes and solutions:

- Low protein load. Increase the amount of protein load. Be sure to have at least 1-5 ng protein on the gel.
- Some proteins may need longer fixing time. Increase the time for fixing the gel to 2 hours or overnight.

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This is probably keratin contamination from fingertips or airborne sources. We recommend wearing gloves at all times during electrophoresis and staining steps, and rinsing the gel wells with ultrapure water or running buffer before sample loading.

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Here are possible causes and solutions:

- Stopper not added to the gel at the appropriate time. Be sure to add the stopper slightly before the desired stain intensity is reached.
- Protein is overloaded. Decrease the protein load on the gel.

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

Here are possible causes and solutions:

- Loss of silver ions from the gel. Limit the wash after staining to 30-60 seconds.
- Stainer or developer solution not prepared properly. Make sure that the solutions are prepared correctly using ultrapure water.
- Low protein load. Increase the amount of protein load. Be sure to have at least 1-5 ng protein on the gel.

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

Here are possible causes and solutions:

- Poor water quality. Use ultrapure water of >18 megohm/cm resistance for preparing solutions or rinsing.
- Staining trays not clean or containing solutions left over from prior silver staining. Use staining trays dedicated for silver staining. After silver staining, wash trays with soap and water, and rinse them with ultrapure water.
- Improper washing done between steps. Do not skip or reduce any washing steps.
- Gels are bent or torn. Be careful during handling of the gel. Remove the gels carefully from the cassette after electrophoresis making sure that the gels do not tear.
- Gels are not completely submerged during staining. Be sure to completely immerse gels in staining solution and perform all steps using a rotary shaker for even staining.

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

Here are possible causes and solutions:

- High concentration of DTT (>50 mM) in the sample. Use 30-50 mM DTT for reducing the sample.
- To prevent streaking, reduce and alkylate the sample as follows: Reduce the sample with freshly prepared DTT to a final concentration of 17 mM and heat the sample at 70 degrees C for 10 minutes. Then, alkylate the sample with freshly prepared iodoacetamide to a final concentration of 35 mM and heat the sample at 70 degrees C for 10 minutes. Add SDS sample buffer without DTT to the reduced and alkylated sample and proceed with electrophoresis and silver staining.
- Presence of thioflavin in the sensitizer. We recommend heating the 30% ethanol wash in the microwave oven before adding it to the gel after the sensitizing step, and also washing the gel with water a bit longer. You can try this with the turbo method but you risk losing the effects of the sensitizer.

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

The kit should be able to detect greater than or equal to 0.3 ng of protein.

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Glutamic acid, aspartic acid, and cysteine thiols are the most reactive with the SilverXpress and SilverQuest staining methods.

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Yes this is possible. We recommend destaining the gel with water (it is not necessary to remove all the stain, but if you would like to do so, we recommend soaking the gel in 50% ethanol followed by numerous water washes). If the SimplyBlue stained gel was destained using salt, we recommend washing the gel numerous times in water to remove all the salts before proceeding with the Silver staining protocol. Further, we recommend beginning the silver staining protocol with the fix step (this will help to remove any methanol/ethanol and salts from the previous staining).

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We recommend using the Mark12 Unstained Standard with the SilverXpress and SilverQuest Silver Staining kits. For the SilverQuest Silver Staining kit, we recommend diluting the Mark12 Unstained Standard to 1:10 and loading 5 µL per lane. For the SilverXpress Silver Staining kit, we recommend diluting the Mark12 Unstained Standard to 1:20 and loading 5 µL per lane.

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We recommend storing the SilverQuest Silver Staining kit at room temperature and the SilverXpress Silver Staining kit at 4 degrees C. Both kits are stable for one year from date of shipment when properly stored.

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