Bolt™ Welcome Pack A (4-12%, 10-well), 1 Welcome Pk. kit - FAQs

•降低电压、电流或缩短转印时间
•确保转膜缓冲液的甲醇浓度合适；可使用浓度为10–20%的甲醇，从而去除SDS-蛋白质复合物中的SDS，并促进蛋白质与膜的结合。
•确保转膜缓冲液的SDS浓度合适（若加入了SDS），SDS浓度不要超过0.02–0.04%。过多的SDS会阻碍蛋白质与膜的结合。过多的SDS会阻碍蛋白质与膜的结合。
•检查膜的孔径和靶标蛋白质的大小。小于10kDa的蛋白质很容易穿过0.45μm孔径的膜。如果您的目标蛋白质小于10 kDa，那么最好使用0.2μm孔径的膜。

•增加电压、电流或转印时间
•凝胶和SDS-蛋白质复合物中的SDS会促进蛋白质从凝胶中洗脱，但抑制蛋白质与膜的结合。这种抑制作用在硝化纤维素膜上的强度大于PVDF膜。对于难以从凝胶中洗脱的蛋白质，如大分子量蛋白质，可在转膜缓冲液中加入少量SDS以改善转印效果。我们建议在组装三明治前将凝胶置于含0.02–0.04% SDS的2x转膜缓冲液（无甲醇）中预平衡10分钟，然后使用含10%甲醇和0.01% SDS的1X转膜缓冲液进行转印。
•甲醇可去除SDS-蛋白质复合物中的SDS，促进蛋白质与膜的结合，但对凝胶本身有一些不良影响，会降低转印效率。甲醇可能导致孔径减小、某些蛋白质发生沉淀以及一些碱性蛋白质带正电荷或变为中性。应确保转膜缓冲液的甲醇浓度不高于10–20%，并使用高质量的分析级甲醇。

预染标准品具有与每种蛋白质共价结合的染料，这将导致标准品在不同的缓冲系统（即不同的凝胶）中迁移率不同。因此，使用预染标准品进行分子量估算将仅得出蛋白质的表观分子量。预染标准品可用于分子量估算、确认凝胶迁移和估算转膜效率，但对于需要精确估算分子量的应用，应使用非预染标准品。

•上样时，请注意确保相邻样品泳道没有交叉污染。
•确保每个泳道上标准品的量都是正确的。蛋白质上样过多会导致产生额外的条带，这个问题在使用银染凝胶时尤为突出。
•标准品储存不当或反复冻融会导致蛋白质降解。

建议如下：

•确保每个泳道上标准品的量都是正确的。蛋白质上样过多会导致模糊，这个问题在使用银染凝胶时尤为突出。
•条带在低百分比凝胶中不能很好地分辨。尝试使用更高百分比的凝胶。
•如果在转膜/检测后条带看起来不明显和模糊，可能是由于抗体浓度过高。遵循制造商建议的稀释度或通过斑点印迹确定最适抗体浓度。

建议如下：

•检查使用的凝胶类型/凝胶百分比。可能会由于凝胶类型和/或百分比的不同而不能看到所有条带。例如，蛋白质标准品的最小条带可能不能在非常低百分比的凝胶上分辨，而较高分子量条带可能不能在高百分比凝胶上分辨。
•检查蛋白质分子量标准品的有效日期。由于蛋白质降解，过期批次可能导致条带褪色或缺失。
•检查蛋白质分子量标准品的储存条件。不适当的储存条件会损害标准品中蛋白质的稳定性。
•确保蛋白质分子量标准品在上样到凝胶上之前未加热/煮沸。我们的蛋白质分子量标准品可直接上样，我们不建议将其加热/煮沸，因为这可能会导致标准品中的蛋白质降解。

电流异常升高的最常见原因是转膜缓冲液。如果转膜缓冲液浓度太高，会导致电导率增加和电流升高。如果不小心用Tris-HCl代替了转膜缓冲液所需的Tris base，也会导致高电流。Tris-HCl可使缓冲液pH降低，引起电导率和电流升高，从而导致过热。我们建议检查转膜缓冲液及其试剂成分，然后重新稀释或重新配制缓冲液。

•将Tris-甘氨酸转膜缓冲液的pH增加至9.2，可使pl低于9.2的所有蛋白质朝阳极方向迁移。
•使用Tris-甘氨酸转膜缓冲液，并在凝胶两侧各放一张膜。碱性高于转膜缓冲液pH的蛋白质，将被凝胶阴极侧的膜捕获。随后，可以用相同的方式处理两张膜。
•转印前，将凝胶置于含0.1% SDS的Tris-甘氨酸转膜缓冲液中孵育15分钟。少量的SDS会给予蛋白质足够的电荷，使蛋白质朝阳极端单向移动，并且在大部分情况下不会使蛋白质变性。然后，使用常规Tris-甘氨酸转膜缓冲液进行转印。

对于大于100 kDa的蛋白质，我们建议在组装三明治前，将凝胶置于含有0.02-0.04% SDS的2XNuPAGE转膜缓冲液（无甲醇）中预平衡10分钟，然后使用含甲醇和0.01%SDS的1XNuPAGE转膜缓冲液进行转印。

以下是可能原因和解决方案：

•凝胶与膜之间存在气泡，阻碍了蛋白质转印。应确保用玻璃吸管滚过膜表面，除去凝胶与膜之间的所有气泡。
•使用了过期或有折痕的膜。应使用新的、无破损的膜。

条带呈旋涡状和扩散状通常是因为分子在与膜结合前发生了横向移动。以下是可能原因和解决方案：

- 凝胶与膜接触不良：凝胶应与膜通过毛细管作用粘在一起，因此，应使用玻璃吸管滚过凝胶/膜三明治的每一层表明，使凝胶与膜良好接触。在组装三明治时，使用一次性吸管在每一层多加一点转膜缓冲液，也有助于凝胶与膜的接触。此外，应完全浸透海绵垫（戴上手套，将海绵垫置于转膜缓冲液中并向下压，挤出所有气泡）。
- 对凝胶的压力不足：凝胶/膜三明治必须牢固装在两部分印迹模块之间。尝试多加一个海绵垫或将失去弹性的海绵垫换成新的。
- 过度挤压凝胶：过度挤压的一个明显表现是凝胶过于扁平。在三明治被过度挤压的情况下，应适当移除海绵垫，降低对凝胶和膜施加的过多压力即可合拢转膜模块。

注意：未压缩海绵垫的高度应比密封垫片高0.5–1.0 cm。

以下是可能原因和解决方案：

•转膜缓冲液的离子强度较高。应按照使用手册的说明来配制缓冲液。
•电源运行时的电流接近电源电流极限。应使用具有更高极限的电源。

以下是可能原因和解决方案：

- 转印时间过短：逐渐增加转印时间，每次增加15分钟。
- 凝胶类型不合适：检查所用凝胶的比例，并换成更高比例的凝胶。
- SDS的用量不合适：在转膜缓冲液中加入0.01–0.02% SDS，促进蛋白质迁移出凝胶。
- 甲醇浓度不合适：降低转膜缓冲液中甲醇的浓度。

注意：与中等至低分子量蛋白质相比，高分子量蛋白质通常不能完全转印。

以下是可能原因和解决方案：

- 转印时间过长：逐渐缩短转印时间，每次减少15分钟。
- SDS的用量不合适：不要在转膜缓冲液中加入SDS。
- 甲醇浓度不合适：在转膜缓冲液中额外加入甲醇，以增强膜的结合能力。
- 凝胶类型不合适：检查所用凝胶的比例，并换成更高比例的凝胶。
- 上样量过多：减少上样量。
- 最后，如果使用的是硝化纤维素膜，则换成结合能力更强的PVDF膜。

可能是因为凝胶/膜三明治的组装顺序反了，从而使蛋白质迁移到了缓冲液中。应按照使用手册中的说明，按正确顺序组装转印三明治。

电流异常升高的最常见原因是缓冲液。如果缓冲液浓度太高，会导致电导率增加和电流升高。如果不小心用Tris-HCl代替了转膜缓冲液所需的Tris base，也会导致高电流。Tris-HCl可使缓冲液pH降低，引起电导率和电流升高，从而导致过热。应检查转膜缓冲液及其试剂成分，然后重新稀释或重新配制缓冲液。

以下是可能原因和解决方案：

- 不小心将缓冲液稀释过度，从而增加了电阻，使电导率和电流降低：检查转膜缓冲液及其试剂成分，然后重新稀释或重新配制。
- 电路损坏或故障，例如电极腐蚀或损坏，或电源故障：检查设备。
- 转印模块泄漏（表现为电流迅速下降和模块中缓冲液体积迅速减少）：确保内层缓冲液槽中的缓冲液足以浸没转膜模块。
- 未移除凝胶盒底部的胶带：再次确认已移除凝胶底部的胶带。

DTT is not stable, so it must be added and the reduction performed just prior to loading your samples.

Find additional tips, troubleshooting help, and resources within our Protein Gel 1D Electrophoresis Support Center.

Precipitation of the LDS or SDS at 4 degrees C is normal. Bring the buffer to room temperature and mix until the LDS/SDS goes into solution. If you do not want to wait for it to dissolve, you can store the sample buffer at room temperature.

Find additional tips, troubleshooting help, and resources within our Protein Gel 1D Electrophoresis Support Center.

Similar to NuPAGE gels with storage temperatures of 4 to 25 degrees C.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

- Decrease voltage, current or length of transfer time
- Make sure that the methanol concentration in the transfer buffer is proper; use a methanol concentration of 10-20% methanol removes the SDS from SDS-protein complexes and improves the binding of protein to the membrane.
- Make sure that the SDS concentration (if added) in the transfer buffer is proper, don't use more than 0.02-0.04% SDS. Using too much SDS can prevent binding of proteins to the membrane.
- Check the pore size of the membrane and the size of the target protein. Proteins smaller than 10 kDa will easily pass through a 0.45 µm pore size membrane. If proteins smaller than 10 kDa are of interest, it would be better to use a 0.2 µm pore size membrane.

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

- Increase voltage, current or length of time for transfer
- SDS in the gel and in the SDS-protein complexes promotes elution of the protein from the gels but inhibits binding of the protein to membranes. This inhibition is higher for nitrocellulose than for PVDF. For proteins that are difficult to elute from the gel such as large molecular weight proteins, a small amount of SDS may be added to the transfer buffer to improve transfer. We recommend pre-equilibrating the gel in 2X Transfer buffer (without methanol) containing 0.02-0.04% SDS for 10 minutes before assembling the sandwich and then transferring using 1X transfer buffer containing 10% methanol and 0.01%SDS.
- Methanol removes the SDS from SDS-protein complexes and improves the binding of protein to the membrane, but has some negative effects on the gel itself, leading to a decrease in transfer efficiency. It may cause a reduction in pore size, precipitation of some proteins, and some basic proteins to become positively charged or neutral. Make sure that the methanol concentration in the transfer buffer is not more than 10-20% and that high-quality, analytical grade methanol is used.

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

Pre-stained standards have a dye that is covalently bound to each protein that will result in the standard migrating differently in different buffer systems (i.e., different gels). As a result, using a pre-stained standard for molecular weight estimation will only give the apparent molecular weight of the protein. Pre-stained standards may be used for molecular weight approximation, confirming gel migration and estimating blotting efficiency but for accurate molecular weight estimation, an unstained standard should be used.

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

- While loading, take care to make sure that there is no cross-contamination from adjacent sample lanes.
- Make sure that the correct amount of standard is loaded per lane. Loading too much protein can result in extra bands and this is a problem especially with silver-stained gels.
- Improper storage of the standard or repeated freeze/thawing can result in protein degradation.

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

Here are some suggestions:

- Make sure that the correct amount of standard is loaded per lane. Loading too much protein can cause smearing and this is a problem especially with silver stained gels.
- Bands will not be as well resolved in low percentage gels. Try using a higher percentage gel.
- If the bands look smeary and non-distinct after a western transfer/detection, this may be due to the antibody being too concentrated. Follow the manufacturer's recommended dilution or determine the optimal antibody concentration by dot-blotting.

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

Here are some suggestions:

- Check the gel type/percentage of the gel that was used. Depending on the gel type and/or percentage, all the bands may not be seen. For example, the smallest bands of the protein standard may not resolve on a very low percentage gel whereas the higher molecular weight bands may not resolve on a high percentage gel.
- Check the expiration date on the protein standard. Expired lots may result in faded or missing bands due to protein degradation.
- Check the storage conditions for the protein standard. Improper storage conditions will compromise the stability of the proteins in the standard.
- Make sure that the protein standard was not heated/boiled prior to loading on the gel. Our protein standards are ready to load and we do not recommend heating/boiling them as this may cause degradation of proteins in the standard.

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

The most common cause of abnormally high current is the transfer buffer. If the transfer buffer is too concentrated, this leads to increased conductivity and current. High current may also occur if Tris-HCl is accidentally substituted for the Tris base required in the transfer buffer. This will again result in low buffer pH and lead to increased conductivity and current and subsequently, overheating. We recommend checking the transfer buffer and its reagent components and re-diluting or remaking the buffer.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

- Increase the pH of Tris-Glycine transfer buffer to 9.2, allowing all the proteins below pI 9.2 to transfer towards the anode electrode.
- Use the Tris-Glycine transfer buffer and place a membrane on both sides of the gel. If there are any proteins that are more basic than the pH of the transfer buffer, they will be captured on the extra membrane placed on the cathode side of the gel. Both membranes can then be developed in the same manner.
- Prior to blotting, incubate the gel for 15 minutes in Tris-Glycine transfer buffer containing 0.1% SDS. The small amount of SDS will give the proteins enough charge to move unidirectionally towards the anode and in most cases, should not denature the protein. Proceed with the transfer using regular Tris-Glycine transfer buffer.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

For proteins larger than 100 kDa, we recommend pre-equilibrating the gel in 2X NuPAGE Transfer buffer (without methanol) containing 0.02-0.04% SDS for 10 minutes before assembling the sandwich and then transferring using 1X NuPAGE transfer buffer containing methanol and 0.01% SDS.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

Here are possible causes and solutions:

- Presence of air bubbles between the gel and the membrane preventing the transfer of proteins. Be sure to remove all air bubbles between the gel and membrane by rolling a glass pipette over the membrane surface.
- Expired or creased membranes used. Use fresh, undamaged membranes.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

The swirling and diffuse banding patterns are typical of molecules moving laterally before binding to the membrane during transfer. Here are possible causes and solutions:

- Poor contact between the gel and the membrane: The gel should be attached to the membrane through capillary action. To ensure that this happens, make sure that you roll over the surface of each layer of the gel/membrane sandwich with a glass pipette to ensure good contact between the gel and the membrane. It is helpful to use a disposable pipette to place some extra transfer buffer on the surface of each layer as the sandwich is being made. Also, the pads need to be fully saturated (push down with gloved hand when they are placed in transfer buffer to make sure there are no air bubbles.)
- Under-compression of the gel: The gel/membrane assembly should be held securely between the two halves of the blot module. Try adding another pad or replace any pads that have lost their resiliency with fresh ones.
- Over-compression of the gel: A good indication of over-compression is if the gel has been excessively flattened. In the event that the sandwich is over-compressed, remove enough pads so that the blotter can be closed without exerting excess pressure on the gel and membrane.
Note: The height of the uncompressed pads should be 0.5-1.0 cm above the level of the sealing gasket.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

Here are possible causes and solutions:

- High ionic strength of the transfer buffer. Prepare the buffer as described in the manual.
- Power supply is operating at a current close to the current limit of the power supply. Use a power supply with higher limits.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

Here are possible causes and solutions:

- Too short a transfer time: Increase the blotting time by 15 minute increments.
- Inappropriate gel type: Check the percentage of the gel used and switch to a higher percentage gel.
- Inappropriate amount of SDS: Add 0.01-0.02% SDS to the transfer buffer to facilitate migration of the protein out of the gel.
- Inappropriate methanol content: Decrease the amount of methanol in the transfer buffer.
Note: Higher molecular weight proteins usually do not transfer completely as compared to mid to low molecular weight proteins.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

Here are possible causes and solutions:

- Too long a transfer tim: Shorten the transfer time by 15 minute increments.
- Inappropriate amount of SDS: Do not include any SDS in the transfer buffer.
- Inappropriate methanol content: Add additional methanol to the transfer buffer to increase the binding capacity of the membrane.
- Inappropriate gel type: Check the percentage of the gel used and switch to a higher percentage gel.
- Sample overloaded: Decrease the sample load.
- Finally, if using nitrocellulose membrane, switch to PVDF which has a higher binding capacity.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

It is possible that the gel/membrane sandwich was assembled in the reverse direction such that the proteins have migrated out into the buffer. Assemble the blot sandwich in the correct order using instructions provided in the manual.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

The most common cause of abnormally high current is the buffer. If the buffer is too concentrated, this leads to increased conductivity and higher current. High current may also occur if Tris-HCl was accidentally substituted for the Tris base required in the transfer buffer. Tris-HCl results in a low buffer pH and leads to increased conductivity and current, and, subsequently, overheating. Check the transfer buffer and its reagent components, re-dilute, or remake the buffer.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

Here are possible causes and solutions:

- The buffer was accidentally made too dilute, therefore increasing resistance and thus lowering conductivity and current: Check the transfer buffer and its reagent components and then re-dilute it or remake it.
- The circuit is broken or impeded, as in the case of a corroded or broken electrode or malfunctioning power supply: Check the equipment.
- There is a leak in the blot module (this is indicated by a drastic decrease in current and in buffer volume within the module): Ensure that the inner buffer chamber is filled sufficiently so that the wells are covered with buffer.
- Tape at the bottom of the gel cassette was not removed: Double check that the tape on the bottom of the gel has been removed.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.