Mini Blot Module - FAQs

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。

我们建议转印时间均为60分钟，硝化纤维素膜使用10 V电压，PVDF膜使用20V电压。另一种更快速的转印条件为电压15V、转印时间30-45分钟，但灵敏度可能会轻微降低。

我们建议使用含10%甲醇的1x Bolt转膜缓冲液。

不需要。但是，我们建议在小型凝胶电泳槽中加水，以疏散转印过程中产生的热量。

通常，每个小型转印模块需要200–250 mL 1X转膜缓冲液，比其他转印设备少2-4倍。

小型转印模块专为小型凝胶电泳槽设计。该模块也适用于Bolt小型凝胶电泳槽（2014年12月31日起停产），但不适用于XCell SureLock Mini Cell或其他供应商生产的电泳槽。

由于通用的电极设计，小型转印模块可装在小型凝胶电泳槽的任意一侧。

我们建议使用1个转印模块转印1块凝胶。

我们建议一开始使用2块海绵垫。如果海绵垫随着使用而逐渐变薄，可增加使用数量。

以下是我们可提供的小型转印模块替换部件：

•切胶刀，货号EI9010
•Bolt电泳槽基座，货号B4478640
•凝胶电泳槽，货号B4478641
•Bolt电泳槽盖，货号B4478591
•凝胶盒夹，右，货号B4478592
•凝胶盒夹，左，货号B4478593
•电源适配器，货号ZA10001

我们建议在使用完毕后用去离子水清洗转印模块。为了清除转印模块中所有的残余沉积物，可使用含50%硝酸的去离子水清洗转印模块内部区域，直至去除残余沉积物。去除沉积物后，使用去离子水清洗模块至少3次。不要将转印模块在硝酸中浸没或浸泡过夜。
注意：制备硝酸溶液时，应戴手套。

是的，我们提供小型转印模块（货号B1000），适用于小型凝胶电泳槽。该转印模块也可用于Bolt小型凝胶电泳槽（2014年12月31日起停产）。请注意，Bolt小型转印模块（2014年12月31日起停产）对Bolt小型凝胶电泳槽和小型凝胶电泳槽均适用。

Bolt Bis-Tris Plus凝胶也可使用XCell SureLock Mini Cell、iBlot干转系统或Invitrogen半干转仪进行转印。

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.

The shelf life for Bolt gels is similar to that for NuPAGE gels: 12 months from the date of shipment.

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

While they are both Bis-Tris based gels, the chemistries are very different since Bolt gels are optimized for western blotting. Another key difference is the wedge well design of the Bolt gels, which allows larger sample volumes to be loaded.

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

The most common power supplies from Thermo Fisher Scientific, Bio-Rad, and Hoefer are compatible. Also, power supply adapters are available for power supplies not designed for use with covered or non-retractable power leads. Thermo Fisher Owl branded gel tanks are not compatible

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

Yes. While we would prefer that you use our devices, Bolt gels can also be transferred using devices from Bio-Rad, including the Mini Trans-Blot Cell, Trans-Blot SD Semi-Dry Transfer Cell, or Trans-Blot Turbo Transfer System.

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

Yes. You can use the iBlot Dry Transfer System or XCell II Blot Module with SureLock tank and achieve similar transfer efficiency.

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 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.

We recommend transferring for 60 minutes at 10 volts with nitrocellulose and 20 volts with PVDF. An optional faster transfer for 30-45 minutes at 15 volts can be used, but a slight loss of sensitivity may be observed.

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

We recommend using 1x Bolt Transfer buffer with 10% methanol.

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

No. However, we recommend adding water to the Mini Gel Tank to help dissipate any heat that may be generated during transfer.

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

Typically, 200-250 mL of 1X transfer buffer is needed per module which is 2-4 times less than with other transfer devices.

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

The Mini Blot Module is designed exclusively for the Mini Gel Tank. It will also fit in the Bolt Mini Gel Tank (discontinued as of December 31, 2014) but will not fit in the XCell SureLock Mini Cell or other vendors' electrophoresis tanks.

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

Due to the universal electrode design, the Mini Blot Module fits on either side of the Mini Gel Tank.

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

We recommend transferring a single gel per blot module.

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

We recommend using 2 sponge pads to start with. If the sponge pads become thinner with extended use, then an additional sponge pad can be used.

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

Here are the replacement parts we offer for the Mini Blot Module:

- Mini Blot Module Gasket, Cat. No. B1001
- Gel Knife, Cat. No. EI9010
- Bolt Tank Base, Cat. No. B4478640
- Gel Runner Tank, Cat. No. B4478641
- Mini Gel Tank Lid, Cat. No. A25944
- Cassette Clamp, Right, Cat. No. B4478592
- Cassette Clamp, Left, Cat. No. B4478593
- Power Supply Adapters, Cat. No. ZA10001

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

We recommend rinsing the blot module with deionized water after use. To clean any residual build-up in the blot module, apply 50% nitric acid in deionized water to areas inside the blot module until residual build-up is removed. Once the build-up is removed, rinse the module at least three times in deionized water. Do not submerge the blot module or soak overnight in nitric acid.

Note: Use gloves when preparing the nitric acid solution.

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

Yes, we offer the Mini Blot Module (Cat. No. B1000), designed to be used with the Mini Gel Tank. This blot module will also work with the Bolt Mini Gel Tank (discontinued as of December 31, 2014). Please note that the Bolt Mini Blot Module (discontinued as of December 31, 2014) is also compatible with both the Bolt Mini Gel Tank and the Mini Gel Tank.

Bolt Bis-Tris Plus gels can also be transferred using the XCell SureLock Mini Cell, iBlot Dry transfer system, or using the Invitrogen Semi-Dry Blotter.

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