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View additional product information for SilverXpress™ Silver Staining Kit - FAQs (LC6100)
56 product FAQs found
是的，您可以使用溴化乙锭、SYBR Green I、SYBR Green II和SilverXpress银染试剂盒对TBE凝胶染色。采用溴化乙锭染色时，将胶侵泡在使用超纯水配制的2 µg/mL溴化乙锭溶液中20分钟。染色后使用超纯水连续冲洗3次，每次10分钟，除去未结合染料。在紫外灯下观察条带。
以下是可能原因和解决方案：
•未在适当时间将终止液加到凝胶上。加入终止试剂的时间应稍早于达到所需条带染色强度的时间。
•蛋白上样量过多。降低凝胶上的蛋白上样量。
这通常是因为蛋白上样量过多。我们建议降低每条条带的蛋白上样量。对于未知蛋白，为了确定最佳上样量，可能需要进行连续稀释。
使用以下视觉线索作为正确完成每一步的标志：
•敏化步骤完成后，低比例浓缩胶与分离胶相比出现白色不透明物[状]，表明这一步正确完成。
•敏化步骤后第一次水洗时，小型凝胶卷曲成圆柱形并漂在液面。
•同时加入Stainer A和Stainer B时，形成短暂可见的棕色沉淀。这种棕色沉淀短暂出现后消失，表明染色液正确混合。如果棕色不褪去，则丢弃溶液，并在干净的玻璃器皿中重新配置混合液。
通常，Tricine凝胶的背景染色会稍高于Tris-甘氨酸凝胶。与Tris-甘氨酸凝胶相比，Tricine凝胶中相对较高的溶质浓度可能会减慢溶液进入凝胶的速度。为解决该问题，可延长敏化步骤中的浸泡时间（可过夜），然后再继续后续步骤。
可尝试使用硫代硫酸盐（也被称为“法梅减薄液”）降低背景。但是，它也会使条带脱色，所以，应稀释浓度。若凝胶在终止液中的浸泡时间超过推荐时间，同样也会降低背景和条带染色强度。
以下是可能原因和解决方案：
•蛋白上样量较低。增加蛋白上样量。应确保蛋白上样量至少为1-5 ng。
•水质较差。应使用电阻率大于18 megohm/cm的超纯水制备溶液或冲洗。
•润洗时，所用水的体积不正确。所有成分都应精确量取，并严格遵守实验方案。
•染色液或显色液的制备不恰当。假设上样量充足，则染色失败最可能是因为银染液或显色液的制备不恰当。如果加入显色液后5分钟未观察到条带，我们建议在染色托盘中直接加入5 mL显色液。应确保使用超纯水正确制备溶液。
如果溶液暂时变为棕色，然后又变透明，则属于正常情况（有时该过程非常快，观察不到）。但是，如果溶液保持为棕色，则表示用于混合染色剂的瓶子中存在污染物，通常是因为瓶中含有之前使用的溶液。
不能，凝胶过度染色是不可逆转的。如果将凝胶放在显色液中，凝胶颜色会持续加深，并在30分钟内变成黑色。所以，必须小心监测显色过程，并适时加入终止试剂。由于溶液是瞬时渗入凝胶的，所以加入终止试剂的时间可能需要稍早于达到所需条带染色强度的时间。特别是在蛋白上样量高的凝胶中，显色非常迅速。
如果怀疑因不完全固定而丢失蛋白，可延长浸泡间隔时间。但是，过夜固定会削弱染色性能。如果不小心大量延长了固定时间，建议在显色后增加洗涤步骤，从而将后续干燥过程中发生凝胶破裂的可能性降至最低。
以下是可能原因和解决方案：
- 水质较差。应使用电阻率大于18 megohm/cm的超纯水制备溶液或冲洗。
- 染色托盘不干净或含有上次银染残留的溶液。使用银染专用托盘。银染后，用肥皂和水洗涤托盘，并用超纯水冲洗干净。
- 不同步骤之间的洗涤处理不恰当。不要跳过或减少任何洗涤步骤。
- 凝胶弯曲或撕裂。处理凝胶时应小心。电泳后，小心将凝胶从凝胶盒中取出，不要撕裂凝胶。
- 染色时，凝胶未完全浸没。应确保凝胶完全浸没在染色液中，所有步骤均使用旋转式摇床以使得染色更加均匀。
这可能是指尖或空气中的角蛋白污染物。我们建议在电泳和染色步骤中全程佩戴手套，并在上样前用超纯水或电泳缓冲液冲洗凝胶孔。
以下是可能原因和解决方案：
•上样孔中的污染物进入凝胶。上样前，使用1X电泳缓冲液冲洗上样孔5次或更多。
•水质较差。应使用电阻率大于18 megohm/cm的超纯水制备溶液或冲洗。
•制备试剂所用的设备污染——使用玻璃棒和灭菌移液管制备试剂。彻底洗涤玻璃器皿。
冷冻的SilverXpress银染试剂盒应该可以使用；之前的测试表明，冷冻的试剂盒性能不变。
可以，由于敏化溶液不含戊二醛，您可在SilverQuest染色凝胶完全脱色后，将蛋白质转印到膜上。
SilverQuest银染试剂盒可兼容质谱（MS）分析，而SilverXpress银染试剂盒不能兼容质谱分析。
我们推荐将凝胶在第二次敏化洗涤溶液中保存过夜。尽管一些其他试剂盒建议将凝胶保存在固定步骤，但我们发现固定过夜有损染色性能。
SilverXpress银染试剂盒可达到的灵敏度水平主要取决于蛋白质的类型和分离程度。通常，非还原型样品可在次纳克范围内产生极好的结果。SilverXpress试剂盒可检测到0.86 ng非还原型BSA，而标准Coomassie染色只能检测到50 ng以上的相同类型蛋白质。如果您使用了Mark12标准品，1:20的稀释倍数适合作为对照。还原型蛋白质所得结果的灵敏度较低。如果使用BME作为还原剂，则可能获得与非还原型样品相等的灵敏度。然而，如果使用DTT作为还原剂，则预期灵敏度在纳克范围，而非次纳克范围。
我们通过对银染法进行轻微改进，得到了良好的结果。我们建议跳过固定步骤，并将敏化溶液的成分更改为2 mL敏化剂加198 mL超纯水。这种改进可将灵敏度变为0.3 ng检测含有50个碱基的核酸，而常规方案的灵敏度为检测0.9 ng含有50个碱基的核酸，溴化乙锭的灵敏度约为检测10 ng含有50个碱基的核酸。此外，为了提高灵敏度，我们推荐将Stainer A的使用量由5 mL增加至7.5 mL，或省略第二个洗涤步骤（该步骤会增强背景）。
不一定，以下两种方法均经过了内部测试：同时加入三种成分，以及混合2种染色剂后再加入水。两种方法可产生相同的结果。
有，可将凝胶在第二次敏化溶液中保存过夜。尽管一些其他试剂盒建议将凝胶保存在固定步骤，但我们发现固定过夜会有损染色性能。
如果没有足够时间连续完成染色操作，可将凝胶在固定液中保存过夜。在某些情况下，延长固定时间可能改善灵敏度和背景染色。
可以，可在染色前24小时内配制SilverXpress染色液。
注意：固定液可保存1个月。如果溶液变为粉色，则需要重新配制。
谷氨酸、天冬氨酸和半胱氨酸硫醇对SilverXpress和SilverQuest银染方法的反应性最强。
这是可以实现的。我们推荐用水脱色（不需要去除所有染料，但如果您想去除所有染料，我们推荐用50%乙醇浸泡凝胶，然后用大量清水洗涤）。如果使用盐对经过SimplyBlue染色的凝胶进行脱色，我们建议用水清洗多次以除去所有盐，然后再进行银染操作。此外，我们推荐从固定步骤开始银染操作（这有助于除去之前染色留下的甲醇/乙醇和盐）。
使用SilverXpress和SilverQuest银染试剂盒时，我们推荐使用Mark12未染色标准品。对于SilverQuest银染试剂盒，我们推荐按1:10稀释Mark12未染色标准品，每个泳道上样5 μL。对于SilverXpress银染试剂盒，我们推荐按1:20稀释Mark12未染色标准品，每个泳道上样5 μL。
我们推荐将SilverQuest银染试剂盒保存于室温，将SilverXpress银染试剂盒保存于4℃。两种试剂盒在合适的保存条件下均可自收货起稳定保存1年。
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.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
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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