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查看更多产品信息 SilverQuest™ Silver Staining Kit - FAQs (LC6070)
27 个常见问题解答
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。
以下是可能原因和解决方案：
•蛋白上样量过低。增加蛋白上样量。上样量应至少为1-5 ng。
•有些蛋白质可能需要较长的固定时间。将凝胶的固定时间延长至2小时或过夜。
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：
•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。
以下是可能原因和解决方案：
•凝胶中的银离子流失。将染色后的洗涤时间限制在30-60秒内。
•染色液或显色液的配制不恰当。应使用超纯水正确配制溶液。
•蛋白上样量过低。增加蛋白上样量。上样量应至少为1-5 ng。
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获得均匀染色。
以下是可能原因和解决方案：
•样品中含有高浓度的DTT（>50 mM）。使用30–50 mM DTT还原样品。
•为防止出现拖尾现象，按以下步骤将样品还原和烷基化：使用新配制的DTT（终浓度为17 mM）还原样品，并在70℃加热样品10分钟。随后，使用新配制的碘乙酰胺（终浓度为35 mM）将样品烷基化，并在70℃加热样品10分钟。在还原和烷基化后的样品中加入不含DTT的SDS样品缓冲液，随后进行电泳和银染。
•敏化剂中含有硫黄素。我们建议在敏化步骤后，先将30%乙醇洗液在微波炉中加热，然后再加到凝胶中，并稍微延长水洗时间。您可在使用turbo方法时尝试上述操作，但是存在损失敏化剂效果的风险。
该试剂盒可检测到≥0.3 ng的蛋白质。
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。
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.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
Glutamic acid, aspartic acid, and cysteine thiols are the most reactive with the SilverXpress and SilverQuest staining methods.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
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).
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
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.