SilverXpress™ Silver Staining Kit - FAQs

Yes, you can stain your TBE gels with ethidium bromide, SYBR Green I, SYBR Green II, and the SilverXpress Silver Staining Kit. For ethidium bromide staining, soak the gel in a 2 µg/mL solution of ethidium bromide in ultrapure water for 20 minutes. Destain by rinsing with three successive 10-minute rinses of ultrapure water. Visualize bands under UV light.

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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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.

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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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Use the following visual cues as landmarks of a properly completed step:

- The low-percentage stacking gel appears whitish opaque as compared to the separating gel after the Sensitizing Step indicating that this step was performed correctly.
- Mini-gels curl up into a cylinder and float on the surface during the first water wash after the Sensitizing Step.
- When Stainer A and Stainer B are added together, a brown precipitate is formed, which is visible only momentarily. This brown “flash” is a good indicator that the staining solutions are mixed correctly. If the brown color does not revert to clear, discard the solutions, obtain clean glassware, and remix the solutions.

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In general, background staining in Tricine gels is slightly higher than in Tris-Glycine gels. The relatively high concentration of solutes in Tricine gels as compared to their Tris-Glycine counterparts appears to slow the rate of solution exchange into the gel. This can be counteracted by increasing the soak time in the sensitization step (you may leave it in overnight) as per the modified procedure, and then proceeding.

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Thiosulfate, known as “Farmer's Reducer”, may be used to attempt to decrease the background. However, it destains the bands as well, so the concentration should be diluted. Leaving the gel in stop solution longer than recommended will decrease background and band intensity, as well.

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Here are possible causes and solutions:

- Low protein load. Increase the amount of protein loaded. Be sure to have at least of 1-5 ng protein on the gel.
- Poor water quality. Use ultrapure water of >18 megohm/cm resistance for preparing solutions or rinsing.
- Incorrect volumes of water used for rinses. Use exact volumes of all components and strictly adhere to the protocol.
- Stainer or developer solution not prepared properly. Assuming that the sample load is sufficient, the most likely cause for staining failure is improper preparation of either the silver staining solution or the developing solution. If no bands are observed within 5 minutes of adding the Developing solution, we recommend adding 5 mL of the Developer directly to the staining tray. Make sure that the solutions are prepared correctly using ultrapure water.

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If the solution turns brown momentarily and then turns clear, this is normal (sometimes this happens so quickly, that this process is not observed). However, if the solution remains brown, this is an indication of a contaminant in the cylinders used to mix the stains, usually introduced by using the same cylinder that contained a prior solution.

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No, it is not possible to reverse the process if the gel is overstained. If left in the Developing solution, the gel will continue to darken and then turn black within 30 minutes. It is therefore very important to carefully monitor the development process and add the Stopper reagent at the appropriate time. Since penetration of solutions into the gel is not instantaneous, it might be necessary to add the Stopper reagent slightly before the desired band intensity has been attained. This is especially the case in heavily loaded gels where developing proceeds very rapidly.

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The length of the soaking interval can be extended if there is suspicion of protein loss through incomplete fixation. However, overnight fixation diminishes stain performance. If fixation times are significantly extended accidentally, then additional post-development washes are recommended to minimize gel cracking during subsequent drying.

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Here are possible causes and solutions:

- Poor water quality. Use ultrapure water of more than 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.

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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:

- Contaminants from the sample wells entered the gel. Carefully rinse the sample wells with 5 or more changes of 1X running buffer prior to sample loading.
- Poor water quality. Use ultrapure water of >18 megohm/cm resistance for preparing solutions or rinsing.
- Contaminated equipment used to prepare reagents - Use glass columns and sterile pipettes to prepare reagents. Wash glassware thoroughly.

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The frozen SilverXpress Staining Kit should be fine to use; frozen kits have been tested in the past and have shown to give an equivalent performance.

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Yes, due to the lack of glutaraldehyde in the sensitizing solution, you should be able to transfer the proteins to a membrane after completely destaining the SilverQuest stained gel.

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The SilverQuest Silver Staining Kit is compatible with mass spectrometry (MS) analysis whereas the SilverXpress Silver Staining kit is not compatible with mass spectrometry analysis.

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We recommend leaving the gel overnight in the second sensitizing wash solution. Although some other kits recommend leaving gels in the fix step, we have found that overnight fixation diminishes staining performance.

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The exact level of sensitivity achievable with the SilverXpress Silver Staining kit depends largely on the type of protein and its preparation. Typically, non-reduced samples yield excellent results in the sub-nanogram range. A 0.86 ng load of non-reduced BSA can be detected with the SilverXpress kit, whereas a standard Coomassie staining procedure can only detect above 50 ng of the same type of protein. If you are using the Mark12 Standard, a 1:20 dilution is an appropriate dilution to use as a control. Reduced proteins may yield less sensitive results. If BME is used as a reducing agent, sensitivities equal to those of non-reduced samples can be expected. However, if, DTT is used as a reducing agent, one can expect sensitivities in the nanogram rather than sub-nanogram range.

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We have used a slight modification that has yielded good results. We recommend skipping the fixation step and changing the composition of the sensitizer solution to 2 mL of sensitizer and 198 mL of ultrapure water. This modification gives 0.3 ng sensitivity down to 50 bases, whereas the normal protocol gives 0.9 ng sensitivity down to 50 bases and ethidium bromide's sensitivity is approximately 10 ng down to 50 bases. Further, to increase sensitivity, we recommend using 7.5 mL of Stainer A instead of 5 mL or eliminate the second wash step (which will also increase background).

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No, both ways have been tested in-house: adding all three ingredients at once and adding the water after combining the two stainers. Both methods give equivalent results.

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Yes, gels can be left overnight in the second Sensitizing Solution. Although some other kits recommend leaving gels in the fix step, we have found that overnight fixation diminishes staining performance.

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The gel can be stored in the fixative overnight if there is not enough time to complete the staining protocol. Longer fixing times may improve the sensitivity and background staining in some cases.

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Yes, the SilverXpress staining solutions can be prepared before staining but not more than 24 hours in advance. Note: The Fixing Solution can be stored for a month. If the solution turns pink, it needs to be remade.

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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.

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