XCell II™ 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。

对于过夜转印，应在冷室中以低功率转印，防止过热。可使用10-15V的恒定电压转印过夜。根据转印效率，对转印条件进行相应调整。

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。

这是完全可以接受的，因为外层溶液槽中的水只起到冷却作用。我们建议在外层溶液槽中加入去离子水，以避免小型电泳槽暴露于甲醇，因为甲醇容易损坏小型电泳槽。

除了海绵垫（货号EI9052），其他组件不单独提供。

我们建议在每次使用完海绵垫以后，用去离子水清洗3-5次，并将水从海绵垫中挤出。

以下是我们可提供的XCell SureLock Mini-Cell替换部件：

适用于XCell SureLock Mini-Cell和XCell II印迹模块的缓冲液挡板：货号 EI0012
适用于XCell II Mini-Cell的SureLock Retrofit试剂盒：货号 EI0020
适用于XCell SureLock Mini-Cell和XCell II Mini-Cell的L-缓冲液槽：货号 EI0013
适用于XCell SureLock Mini-Cell的铂丝（12英寸x 2）：货号 EI9022
XCell SureLock电泳槽盖：货号 EI0010
XCell SureLock楔：货号 EI0011
适用于XCell SureLock Mini-Cell和XCell II印迹模块的缓冲液核心w/电极：货号 EI9014
适用于XCell SureLock Mini-Cell的缓冲液核心维修工具包：货号 EI9021
切胶刀：货号 EI9010
印迹用海绵垫（8）：货号 EI9052

XCell SureLock Mini-Cell包括盖子、凝胶张力楔、下液槽、缓冲液核心、缓冲液挡板和切胶刀。

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.

Here are some options for obtaining more efficient transfer for larger proteins:

1) Pre-equilibrate the gel with 0.02 to 0.04% SDS in 2X transfer buffer without methanol for 10 min before assembling the sandwich.

2) Increase the blotting time incrementally (in 15 min intervals).

3) Add 0.01% or 0.02% SDS to the transfer buffer to help facilitate the migration of the protein out of the gel.

4) Decrease the methanol content in the transfer buffer.

5) Switch to a more appropriate lower-percentage gel. A lower-percentage gel may allow better transfer than a higher-percentage gel.

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

No. The solution placed in the outer chamber serves to dissipate the heat generated during blotting. Water is usually used for this purpose. The recommended transfer conditions generate only a minor heat increase, so it is not necessary to run the unit in an ice bucket or to place it in a cold room. However if you are working with very heat-sensitive proteins, you may wish to do so.

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

Build-up can be removed with 50% nitric acid. Make a solution of 50% nitric acid in deionized water and carefully apply it to areas inside the blot module until residual build-up is removed. Do not submerge the blot module or soak overnight. Use gloves when preparing the solution. Afterwards, rinse the module thoroughly at least three times in fresh deionized water. This treatment should not harm the plastic.

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

The NuPAGE Invitrogen Bis-Tris Gels do not transfer efficiently using a Invitrogen Semi-Dry Blotter as compared to blotting with XCell II Blot Module.
If you decide to use Invitrogen Semi-Dry Blotter for NuPAGE Invitrogen Bis-Tris Gels, use the protocol provided below to ensure efficient transfer of proteins.

1) Prepare 100 mL of 2X NuPAGE Transfer Buffer from 20X NuPAGE Transfer Buffer as follows:
NuPAGE Transfer Buffer (20X) 10.0 mL
NuPAGE Antioxidant (for reduced sample) 0.1 mL
Methanol 10.0 mL
Deionized water 79.9 mL
Total Volume 100 mL
If you are blotting large proteins, please see the Note below.

2) Soak the filter paper and transfer membrane in the transfer buffer.
If you are using Invitrogen pre-cut membrane/filter sandwiches, use three filter papers (0.4 mm/filter in thickness) on each side of the gel or membrane.
If you are not using the Invitrogen pre-cut membrane/filter sandwiches, use two thick filter papers.

3) Assemble the gel/membrane/filter paper sandwich on top of the anode plate as follows:
filter paper
filter paper
filter paper
membrane
gel
filter paper
filter paper
filter paper

4) Perform the transfer at 15 V (constant) for 15 min if you are using the Bio-Rad Trans-Blot Semi-Dry Transfer Cell. For any other semi-dry transfer cell, follow the manufacturer's recommendations.

Note: For transfer of large proteins (>100 kDa), pre-equilibrate the gel in 2X NuPAGE Transfer Buffer (without methanol) containing 0.02-0.04% SDS for 10 min before assembling the sandwich.

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

After staining with SimplyBlue SafeStain, use deionized water for the less strongly retained protein bands on the PVDF membrane.

Increasing methanol or ethanol concentrations up to 70% should destain any remaining bands. You can leave the membrane in the destain indefinitely.

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

There are three common explanations:

1) The buffer was accidentally made too dilute, which increases resistance and lowers conductivity and current. Check the transfer buffer and its reagent components, remake, or redilute.

2) The circuit is broken or impeded, as in the case of a corroded or broken electrode or malfunctioning power supply. Check the equipment.

3) There is a leak in the blot module. This is indicated by a drastic decrease in current and in buffer volume within the module.

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. Three possible explanations are:

1) Poor contact between the gel and the membrane: the gel should contact the membrane across the entire surface for good capillary action. To ensure good contact and remove any bubbles, roll a glass pipette over the surface of each layer of the gel/membrane sandwich. Pipet some extra transfer buffer on the surface of each layer as the sandwich is being made. Also, the blotting pads need to be fully saturated with transfer buffer (push down with a gloved hand when they are submerged to ensure that there are no air bubbles).

2) Undercompression 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 new ones.

3) Overcompression of the gel: A good indication of overcompression is if the gel has been excessively flattened. In the event that the sandwich is overcompressed, remove enough pads so that the blot module 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.

A number of human alkaline phophatases that could be consuming the WesternBreeze substrate have a molecular weight close to 50 kDa. These would be detected as a band of approximately 54 kDa. These proteins are known to dimerize, and can show as a band of approximately 110 kDa in native gels.

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

Less-than-optimal transfer in the second gel is not uncommon and frequently requires adjustments in protocol. To achieve similar transfer efficiencies in the two gels you can transfer them one at a time or consider the following suggestions:

Make sure that the blot module is not overfilled with buffer, i.e., fill to point where pads are just covered and no higher. If the buffer level is too high, some current will bypass the gels. If it is overfilled, shorting, arcing, and other problems can occur.

Use antioxidant when transferring reduced proteins.

Increase transfer time by 30 to 60 minutes. The longer run time allows the slower moving proteins time to move out of the gel.

As proteins are being driven out of the gel by the SDS on their surface, it is important that enough is retained to keep them mobile. However, the methanol that is included in the transfer buffer will remove some of the SDS, and the higher the methanol concentration, the more SDS is washed off of the protein. Methanol is included in order to increase binding to nitrocellulose membranes via hydrophobic interactions. However, if using PVDF or nylon membranes, less or even no methanol may be required. 20% methanol is a good starting point for most situations In the event that the transfer is less than ideal, methanol levels should be reduced by at least 50%. The optimal percentage should be determined empirically. Something to keep in mind is that with increased mobility, the proteins may just move straight though the first membrane. In this case, a second membrane may trap these samples.

It is possible to BRIEFLY (1 min) soak the second gel in transfer buffer with 0.01 to 0.02% SDS before assembling the sandwich. This must be a short soak or the proteins will tend to diffuse out of the gel. Adding 0.01-0.02% SDS to the transfer buffer and using it for the transfer will also aid in protein mobility.

Halfway through the transfer process (30 min), swap the front gel and back gel in the blotter. This is fairly easy to do if you just swap pad-paper-gel-membrane-pad sections.

Another recommendation: if you must transfer simultaneously, and want to be able to compare results of both blots, use an internal control in both gels to get an idea of the relative transfer efficiency and to afford some normalization of the results.

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.

For overnight blotting, perform transfer in the cold room with low power to prevent overheating. Transfer at constant voltage of 10-15 V overnight. Depending on the transfer efficiency, adjust the transfer conditions accordingly.

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. Please see page 17 of the XCell II Blot Module Manual.

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.

This is perfectly acceptable as the water in the outer buffer chamber only serves as a coolant. We recommend adding deionized water to the outer buffer chamber to avoid exposure of the mini-cell to methanol as the mini-cell is susceptible to methanol.

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

Except for the sponge pads (Cat. No. EI9052), none of the other parts are available separately.

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

We recommend cleaning the sponge pads after each use by rinsing with deionized water and squeezing the water out of the sponge pad 3-5 times.

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 XCell SureLock Mini-Cell:
Replacement part - Cat. No.
Buffer Dam for XCell SureLock Mini-Cell and XCell II Blot Module - EI0012
SureLock Retrofit Kit for XCell II Mini-Cell - EI0020
L-Buffer Chamber for XCell SureLock Mini-Cell and XCell II Mini-Cell - EI0013
Platinum Wire for XCell SureLock Mini-Cell (12 inch x 2) - EI9022
XCell SureLock Lid - EI0010
XCell SureLock Wedge - EI0011
Buffer Core w/ Electrodes for XCell SureLock Mini-Cell & XCell II Blot Module - EI9014
Buffer Core Repair Kit for XCell SureLock Mini-Cell - EI9021
Gel Knife - EI9010
Sponge Pad for Blotting (8) - EI9052

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

The XCell SureLock Mini-Cell includes the lid, gel tension wedge, lower buffer chamber, buffer core, buffer dam, and gel knife.

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