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iBlot® Gel Transfer Device - FAQs

查看更多产品信息 iBlot® Gel Transfer Device - FAQs (IB1001)

87 个常见问题解答

我使用一台已经用过2年的iBlot设备进行转印，得到的蛋白质转印结果很不均匀。问题在哪里？

这最有可能是因为转印期间电流分布不均匀，可能是电极太差。我们建议更换新的电极（货号IB1002）。

我正在尝试对我的iBlot进行固件升级，但iBlot在升级过程中冻住了。我该怎么办？

这可能是因为USB连接线在升级过程中断开。请更换电脑和USB连接线再试一次。

我正在尝试对我的iBlot进行固件升级，但是因为升级器一直在寻找iBlot设备而无法进行升级。你们能否帮我排除故障？

iBlot设备必须直接连接到进行固件升级的电脑。不要使用USB转换器。

为了进行环回测试，应首先确认已成功安装驱动和COM端口。
1.在安装了USB串行驱动的情况下，将您的iBlot设备连接到电脑的USB端口。
2.进入Windows设备管理器（开始->控制面板->系统->硬件选项卡->设备管理器）的端口（COM & LTP）选项下，查看是否已成功创建USB串行端口（COM）。如果设备管理器的列表中无COM端口，则尚未成功创建。您可使用http://www.ftdichip.com/Drivers/VCP.htm手动安装驱动。
3.如果有多台设备连接到同一个USB串行端口，您可能需要断开其他设备。

如果上述步骤无效，还有一个可能会解决问题的简单方法是换一根USB连接线。

我订购了iBlot PVDF凝胶转印膜组，但在膜组中找不到PVDF膜。我是否应该要求更换？

PVDF膜可能在处理或运输中丢失。活化的PVDF膜是透明的，很难看到。如果在顶层膜组中没有PVDF膜，我们建议检查阳极膜组（透明托盘）的铝密封片，确认PVDF膜没有粘到密封片上。如果膜粘到了密封片上，我们建议用纯甲醇再次活化膜，并用蒸馏水清洗干净，然后小心放到膜组上。再活化过程不会影响性能。

我使用iBlot干转系统进行转印，发现检测后不同蛋白质的信号强度都很相近。这是为什么？

这最可能是因为蛋白质上样量过多，使检测超出线性范围。由于使用iBlot凝胶转印设备进行干转的免疫检测灵敏度高于半干转或湿转，我们建议您减少蛋白质上样量、使用更稀的抗体或缩短检测时间。您可能需要根据最初的结果进行一些优化。

我使用了iBlot干转系统，但膜上的背景较高。你们有何建议？

这可能是因为使用了TBST缓冲液进行洗涤。我们建议使用PBST或WesternBreeze洗涤液。

我使用了PVDF凝胶转印膜组和iBlot干转系统，但蛋白质未转印到膜上。为什么？

这可能是因为PVDF膜变干或部分变干。PVDF膜的干燥部分比湿润部分颜色更白。应将膜置于100%甲醇中再次活化，并用水清洗，然后再放到转印膜组上。

我使用iBlot干转系统将蛋白质从E-PAGE凝胶上转印出来，结果发现膜上的蛋白质条带是扭曲的。这是为什么？

这是因为上样孔周围的电场不均匀。应使用De-bubbling Roller将E-PAGE凝胶上样孔周围的突出部分压平。为得到最佳转印结果，我们建议对E-PAGE凝胶使用De-bubbling Roller。如果将Blotting Roller用于E-PAGE凝胶，应遵循使用手册（https://tools.thermofisher.com/content/sfs/manuals/iblotsystem_man.pdf）第22页的建议以获得良好的结果。

使用iBlot干转系统时，有些蛋白质穿过了膜。可能原因是什么？

如果转印时间过长，可能发生这种情况。我们建议逐渐缩短转印时间，每次减少30秒。

注意：预染标记物带有电荷，所以穿过膜的可能性比普通蛋白质更大。

使用iBlot干转系统进行蛋白质转印后，通过对凝胶进行染色发现高分子量蛋白质仍留在凝胶中。你们能否帮我排除故障？

如果使用了错误的电压方法或不适当的转印条件，可能会出现这种情况。应按照使用手册（https://tools.thermofisher.com/content/sfs/manuals/iblotsystem_man.pdf）第13页的说明，使用与凝胶类型相符的正确电压方法和运行时间。

对于小型或中型凝胶：
•按照使用手册（https://tools.thermofisher.com/content/sfs/manuals/iblotsystem_man.pdf）第28页的说明，采用乙醇预平衡步骤以改善转印。
•使用较低比例的凝胶来分离高分子量蛋白质。
•逐渐增加转印时间，每次增加30秒。

对于E-PAGE凝胶：
•逐渐增加转印时间，每次增加30秒。
•使用方法P2，转印8分钟。

注意：有些蛋白质残留在凝胶中属于正常情况，因为，与半湿转装置相比，iBlot凝胶转印设备对某些高分子量蛋白质的转印不完全。

使用iBlot干转系统转印后，膜上有很多空白的点。可能原因是什么？

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。应确保使用适用于E-PAGE凝胶的De-bubbling Roller或适用于其他凝胶的Blotting Roller，赶走凝胶与膜之间的所有气泡。
•使用了过期或有折痕的膜。应使用未超过包装有效期的iBlot凝胶转印膜组。

我在使用iBlot干转系统时，没有蛋白质被转印到膜上。可能原因是什么？

这可能是因为没有电流通过或者使用了错误的电压方法。应确保电路闭合，并且有电流通过设备。请确认使用了正确的电压方法（见使用手册（https://tools.thermofisher.com/content/sfs/manuals/iblotsystem_man.pdf）第13页）。

使用iBlot干转系统时，iBlot阳极膜组（底部）转印凝胶熔化成了粘稠的蓝色液体。我该如何防止这种情况再次发生？

若将膜修剪成符合凝胶大小的尺寸，会导致iBlot阴极膜组（顶部）与阳极膜组（底部）直接接触，从而发生熔化情况。必须保持膜的尺寸与转印膜组一致。与膜相比，较小的凝胶尺寸不会影响转印质量。

使用iBlot干转系统时，凝胶周围膜的边缘有时会变成绿色。绿色部分是什么？这是否会影响结果？

变为绿色是由于iBlot转印膜组中的铜离子被液体带走并沉积到膜上。这些沉积物不会影响下游过程。可将染色区域剪掉，但是，对膜进行清洗通常也可除去沉积物。为了将这种作用降至最低，应分别在将滤纸和凝胶放到转印膜组上之前，甩掉多余的水和缓冲液。

使用iBlot干转系统进行凝胶转印后，膜和凝胶都变成蓝色了。这是为什么？

转印时间过长可使铜离子沉积，导致颜色变蓝。应确保每种类型的凝胶都以其相应的推荐时间进行转印。

我使用iBlot干转系统时，发现iBlot阴极膜组（顶部）有一些腐蚀。原因是什么？

这最可能是由顶部膜组的位置不正确引起的。应确保正确放置iBlot阴极膜组（顶部），使铜电极一侧朝上。不要将顶部膜组反向放置。

我的iBlot干转系统显示“错误2”信息。哪里出错了？

以下是可能的原因和解决方案 ：

- iBlot阴极膜组（顶部）接触到了iBlot阳极膜组（底部）上的铜电极： 打开盖子，将iBlot阴极膜组（顶部）向右对齐。轻按一下开始/停止按钮继续运行，或长按开始/停止按钮重新开始运行。
- 各层没有对齐： 按照实验方案中的说明，将各层对齐。确保电极正常连接。
- 电流超过5.5 amp： 选择一个电压较低的程序。打开iBlot盖子，确认膜组对齐。关闭盖子，减去已运行时间后，重新开始运行。将iBlot凝胶转印膜组换成新的。应确保在小型或中型凝胶转印期间使用了iBlot滤纸。

我的iBlot干转系统显示“错误1”信息。哪里出错了？

这表示运行期间出现断路。如果在运行期间打开盖子，会出现这种情况。

盖上盖子，轻按一下开始/停止按钮继续运行，或长按开始/停止按钮重新开始运行。

我在使用iBlot干转系统时，无电流通过（固定好盖子后，红灯没有亮）。你们能否帮我排除故障？

这表明电路未闭合，以下是可能原因和解决方案：

- iBlot一次性海绵遮住了金属接头，或者海绵上的金属接头在左侧： 重新插入iBlot一次性海绵，使海绵上的金属接头位于盖子的右上角，并接触到转印膜组的电极。
- 转印膜组的位置错误或组装不当： 应确保将转印膜组正确放置在转印仪表面，与电极良好接触。确保转印膜组正确安装；首先安装iBlot阳极膜组（底部），然后依次放置凝胶和iBlot阴极膜组（顶部）
- 拉片位置错误： 应确保iBlot阴极膜组（顶部）的拉片在转印仪表面朝向三明治的右侧。
- 将iBlot阳极膜组（底部）放在设备上时，不带有包含电接头的托盘： 组装时，不要拆除iBlot阳极膜组（底部）的托盘。转印依赖于塑料托盘中的底部膜组。
- 将带有托盘的iBlot阴极膜组（顶部）放在设备上： 应该先从红色塑料托盘上取下iBlot阴极膜组（顶部），再放到三明治上。不要使用带有托盘的iBlot阴极膜组（顶部）。
- 盖子折页中的金属安全接头脏了，无法正常连接： 用蘸水的棉签清洁盖子折页中的金属安全接头。

iBlot转印膜块是否兼容Li-COR检测？

兼容，iBlot转印膜组可以兼容Li-COR检测。

iBlot干转系统带有的印迹滚筒是什么？

印迹滚筒（Blotting Roller）是连接有不锈钢把手的塑料滚筒，可用于除去在放置膜块和凝胶期间凝胶与印迹膜之间的所有气泡。

除气泡滚筒是什么？

除气泡滚筒（De-bubbling Roller）是一种不锈钢的铝制滚筒，专用于在安装E-PAGE凝胶转印膜块期间赶走凝胶与印迹膜之间的所有气泡。我们建议仅将除气泡滚筒用于E-PAGE凝胶。若其他类型的凝胶使用该滚筒，可能发生延伸和撕裂。iBlot干转系统带有除气泡滚筒，而iBlot 2干转系统没有。

iBlot E-PAGE拉片是什么？

iBlot E-PAGE拉片是E-PAGE凝胶转印期间使用的一个钢片。将拉片粘贴到iBlot阴极膜块（顶部膜块）上，在E-PAGE凝胶除气泡过程中使用拉片将转印膜块拉向转印表面。

iBlot滤纸的用途是什么？

iBlot滤纸可用于转印小型或中型凝胶。在放置iBlot阴极膜块（顶部膜块）前将滤纸放在凝胶上，可在转印过程中保护凝胶的完整性。iBlot滤纸有两种规格，可为小型和中型凝胶提供有效转印。我们不建议将iBlot滤纸用于E-PAGE凝胶转印。

注意：若小型和中型凝胶转印时不使用iBlot滤纸，可能会使电流超过电流上限，导致电泳期间出错（错误2）。

iBlot干转系统是否有洗脱方案？

传统的洗脱方案是使用0.1 M甘氨酸（pH 2），用于洗脱多克隆抗体。

使用iBlot干转系统时，如何能对大分子量蛋白质实现更好的转印？

分子量大于150 kDa的蛋白质比小分子量蛋白质迁移更慢。因此，将大分子量蛋白质从凝胶转印到膜上需要更长的时间。我们建议使用转印时间为8-10分钟的P3程序来转印这类蛋白，以获得最佳转印结果。

为了提高转印效率，我们还建议在电泳和转印之间增加一个平衡（凝胶浸泡）步骤，并使用NuPAGE Invitrogen 3-8% Tris-acetate凝胶进行电泳。请查看使用 iBlot干转系统转印大分子量和小分子量蛋白质的应用指南（https://www.thermofisher.com/content/dam/LifeTech/migration/en/filelibrary/protein-expression/pdfs.par.87290.file.dat/iblot%20small%20and%20large%20protein%20app%20note.pdf），获取实验方案。

iBlot干转系统转印膜块的铜电极能否回收利用？

为了最大限度地回收iBlot转印膜块的铜网电极，我们已经与美国的一家回收中心达成了协议。为了能够制备可回收的铜电极，请遵循下列说明（https://www.thermofisher.com/content/dam/LifeTech/migration/files/proteins-expression-isolation-analysis/pdfs.par.0358.file.dat/iBlot%20copper%20reycling%20instructions%20NA%20only.pdf）。

我想对小型凝胶（8 x 8 cm）进行蛋白质转印，但我没有iBlot转印膜组（小型）。能否使用iBlot转印膜组（标准）进行小型凝胶转印？

最好不要在标准规格的转印膜组中单独转印一块小量凝胶。尽管在大多数情况下能够实现良好的转印，但转印膜组中未与凝胶直接接触的空余空间可能使膜的整个表面发生扭曲，包括膜上与凝胶接触的部分。如果可能，最好使大于50%的膜与凝胶接触。

iBlot凝胶转印膜块能否冷冻？

iBlot凝胶转印膜块不可以冷冻，因为冷冻会破坏其中含有的凝胶基质。

你们是否提供iBlot的替换用盖锁和电极？

提供，iBlot盖锁的货号为IB1003，iBlot电极（1对）的货号为IB1002。请点击这里（https://tools.thermofisher.com/content/sfs/manuals/iblot_electrode_replacement_man.pdf），查看电极更换说明。

iBlot PVDF凝胶转印膜块中的PVDF膜是预活化的吗？

PVDF膜是预活化的，可立即使用，无需任何乙醇预处理。

iBlot转印膜块的推荐保存条件使什么？

我们建议将iBlot转印膜块保存于室温。

iBlot转印膜块有哪些不同种类？能否单独购买？

以下iBlot转印膜块均可用于iBlot凝胶转印设备，并且可以单独购买：

•iBlot凝胶转印膜块可用于将蛋白质从凝胶转印到硝化纤维素膜或PVDF膜上（蛋白质印迹分析）。硝化纤维素膜或PVDF转印膜组均具有小型和标准规格。
•iBlotDNA转印膜块可用于将DNA从凝胶转印到尼龙膜上（DNA印迹分析）。

进行免疫印迹分析时，哪类凝胶可用于iBlot干转系统？

iBlot干转系统可使用Bolt Bis-Tris Plus、NuPAGE Bis-Tris、Tris-Acetate、Tris-甘氨酸、Tricine（小型和中型凝胶规格）和E-PAGE凝胶。

你们是否仍然提供iBlot凝胶转印设备？

iBlot凝胶转印设备已停产，我们已经推出了升级版iBlot 2凝胶转印设备。iBlot转印膜块、免疫印迹检测膜块和DNA转印膜块仍可购买，但只能用于iBlot凝胶转印设备，不可用于新型iBlot 2凝胶转印设备。仅iBlot 2耗材能够用于iBlot 2凝胶转印设备（https://www.thermofisher.com/order/catalog/product/IB21001）。

iBlot干转系统是否适用于非变性或非变性-蓝色凝胶？

适用，请参考使用iBlot干转系统进行NativePAGE Bis-Tris凝胶免疫印迹分析的应用指南（https://www.thermofisher.com/content/dam/LifeTech/migration/files/proteins-expression-isolation-analysis/pdfs.par.9778.file.dat/co01743%20native%20with%20blot%20app-f.pdf）。

Following the release of the iBlot 2 Gel Transfer Device, are consumables for the original iBlot Gel Transfer Device still available?

Yes, all original iBlot stacks are still available for purchase. You can find them in the original iBlot Gel Transfer Device Reagents and Resources (http://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-assays-analysis/western-blotting/transfer-proteins-western-blot/iblot-dry-blotting-system/reagents-resources-original-iblot-gel-transfer-device.html). However, please note that they are not compatible with the iBlot 2 Gel transfer Device.

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

Is the iBlot 2 Gel Transfer Device compatible with original iBlot Transfer Stacks?

No. Do not use iBlot Transfer Stacks in the iBlot 2 Gel Transfer Device, and do not mix components between iBlot Transfer Stacks and iBlot 2 Transfer Stacks.

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

How can I get better transfer of high-molecular weight proteins?

Proteins larger than ~150 kDa migrate more slowly than smaller proteins. Therefore, more time is required to transfer them from gel to membrane. We recommend extending the transfer time by 8 to 10 minutes for optimal transfer of proteins >150 kDa using the iBlot Dry Blotting System.

To enhance transfer efficiency, we also recommend adding an equilibration (gel-soaking) step between electrophoresis and western transfer and using NuPAGE Invitrogen 3 - 8% Tris-acetate gels for electrophoresis. We have an application note available, titled “Transferring Large and Small Proteins Using the iBlot Dry Blotting System”. To download the PDF, see the iBlot Reagents Resources page. (http://www.thermofisher.com/us/en/home/life-science/protein-expression-and-analysis/western-blotting/western-blot-transfer/iblot-dry-blotting-system/original-iblot-resources.html)

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

Occasionally my western blots have high background. What do you recommend?

This may be a result of insufficient blocking or nonspecific binding. We suggest trying our WesternBreeze Blocker/Diluent (Cat. No. WB7050). We have been using it with good success. Additionally, you should optimize primary and secondary antibody concentrations as generally recommended for any new blotting technique. Many cases of high background can be resolved by further diluting one or both antibody preparations.

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

Is it possible to substitute the membrane from the iBlot Transfer Stack with my specialized membrane?

In theory, you can replace the membrane provided in your iBlot Transfer Stack with any membrane that is compatible with western blotting. To do this, cut the alternative membrane to match the size of your gel, and wet the membrane. Then, either place the alternative membrane on top of the integrated membrane, or carefully remove the integrated membrane from the gel matrix with forceps and replace it with the new membrane. Note that we only support the use of iBlot Transfer Stacks when they are used with the provided instructions.

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

What is the shelf life of nitrocellulose and PVDF iBlot Transfer Stacks?

The minimum guaranteed shelf life of iBlot Transfer Stacks is 2 months. Depending on when you purchase the transfer stack, shelf life will be 2–8 months. The expiration date is printed on the package for each stack.

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

Can iBlot Transfer Stacks be used more than once?

No. The transfer stacks have a finite amount of ions to drive the transfer and are depleted after a single use.

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

Do the PVDF iBlot Transfer Stacks (0.2 µm, non-autofluorescing) require activation prior to use?

No. The PVDF membrane comes preactivated. You just need to open the transfer stack with membrane, place the separation gel on top of the membrane, and apply one layer of moistened filter paper to run (the same as with the nitrocellulose stacks).

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

Sometimes there is green discoloration on the blot around the gel when using the iBlot Dry Blotting System. What is this, and does it affect the results?

The green discoloration is copper deposits from the iBlot Transfer Stacks, and it does not affect the results. To minimize this effect, shake excess water off the filter paper and buffer from the gel before placing each on the stack. The current formulation of stacks minimizes the green discoloration.

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

How can we be more environmentally responsible in regards to the packaging for the iBlot Dry Blotting System?

The plastic in the iBlot Dry Blotting System stack packaging is polyethylene terephthalate (PET) and can be recycled.

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

I want to conduct western transfer with mini gels (8 x 8 cm), but I do not have iBlot Transfer Stacks (Mini). Can I use iBlot Transfer Stacks (Regular) to transfer my mini gels?

It is best not to transfer a single mini gel on a regular-sized transfer stack. Although in most cases the transfer will be fine, empty spaces on the transfer stack that are not in direct contact with a gel could potentially cause distortions across the whole surface of the membrane, including the portion in contact with the gel. It is best to have more than 50% of a membrane in contact with the gel, if possible.

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

Is there a limitation on the thickness of gels that can be used with the iBlot Dry Blotting System?

The iBlot Dry Blotting System has been tested to efficiently transfer protein from gels ranging in thickness from 1 mm to 3 mm. We have not tested gels thicker than 3 mm because they are rarely used for SDS-PAGE.

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

Are Bolt gels compatible with transfer devices from other suppliers?

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.

How can I get better transfer of low molecular weight proteins with the iBlot Dry Blotting System?

Small proteins (under 30 kDa) migrate more rapidly than large ones and hence, need less time to transfer from the matrix of the gel to the membrane. While P3 program for 7 minutes works well with most proteins, less time is needed for the transfer of smaller proteins with the iBlot device. We recommend using P3 program for less than 5-6 mins. Please refer to Transferring Large and Small Proteins Using the iBlot Dry Blotting System Application Note (http://www.thermofisher.com/content/dam/LifeTech/migration/en/filelibrary/protein-expression/pdfs.par.87290.file.dat/iblot%2520small%2520and%2520large%2520protein%2520app%2520note.pdf).

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

How do I destain proteins on a PVDF membrane that were stained with SimplyBlue SafeStain?

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.

I got very uneven transfer of my proteins using an iBlot device that is two years old. What is the issue?

This is most likely due to uneven current distribution during transfer, possible caused by bad electrodes. We recommend getting new electrodes (Cat. No. IB1002).

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

I am trying to do a firmware upgrade for my iBlot and the iBlot froze during the process. How should I proceed?

This could happen due to the USB cable disconnecting during the updating process. Please try again with another computer and a different USB cable.

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

I am trying to do a firmware upgrade for my iBlot and I am unable to proceed as the Updater seems to be endlessly searching for the iBlot device. Can you offer some troubleshooting tips?

The iBlot device must be connected directly to the computer for the firmware upgrade to work. Do not use a USB hub.

In order to make a loop-back test you first need to make sure that the drivers and COM port have been successfully installed.
1. With the USB Serial drivers installed, connect your iBlot device to your computer's USB port.
2. Check in Windows Device Manager (Start -> Control Panel -> System -> Hardware tab -> Device Manager) under Ports (COM & LTP) to see if the USB Serial Port (COM) has been successfully created. If the COM port is not listed in the Device Manager, it has not been successfully created. You can install the driver manually from http://www.ftdichip.com/Drivers/VCP.htm
3. If multiple devices are connected to one USB serial port, you may need to disconnect other devices.

If the above steps don't help, another simple option that may solve the issue would be to try a different USB cable.

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

I ordered an iBlot PVDF Gel Transfer Stack and am unable to find the PVDF membrane in the stack. Should I request a replacement?

It is possible that the PVDF membrane got displaced during handling or shipment. The activated PVDF membrane is transparent, making it difficult to see. If it is not present on top of the stack, we recommend examining the aluminum seal of the Anode Stack (clear tray) to make sure that the PVDF membrane has not adhered to the seal. If the membrane has adhered to the seal, we suggest reactivating the membrane with pure methanol, then rinsing it well in distilled water, and carefully placing it on the stack. The performance will not be affected by the reactivation process.

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

I used the iBlot Dry Blotting System and noticed that the signal intensity was similar for different protein loads after detection. Why is this?

This is most likely due to the protein load being too high, as a result of which detection is not within the linear range. Since the immunodetection sensitivity is higher for dry blotting with the iBlot Gel Transfer Device than for semi-dry or wet blotting, we recommend that you decrease the protein load, use more diluted antibody, or perform detection for shorter time. You may need to perform some optimization based on your initial results.

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

I used the iBlot Dry Blotting System and got high background on the membrane. Can you offer some tips?

This is likely due to use of TBST buffers for washing. We recommend using PBST or WesternBreeze wash solutions.

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

I used PVDF Gel Transfer Stacks with the iBlot Dry Blotting System and my proteins did not transfer to the membrane. What happened?

This is likely due to the PVDF membrane being dry or partially dry. Regions where PVDF membranes are dry appear whiter than places where the membrane is wet. Remove the membrane and reactivate in 100% methanol, and rinse in water before reapplying to the transfer stack.

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

I transferred my proteins from an E-PAGE gel using the iBlot Dry Blotting System and my protein bands are distorted on the membrane. Why is this?

This indicates that a non-uniform electric field was created around the wells. Ensure that the well protrusions on the E-PAGE gel are properly flattened using the De-bubbling Roller. To ensure the best blotting results, we recommend using the De-bubbling Roller with E-PAGE gels. If you use the Blotting Roller with E-PAGE gels, be sure to follow the recommendations on page 22 of the manual (https://tools.thermofisher.com/content/sfs/manuals/iblotsystem_man.pdf) to obtain good results.

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

With the iBlot Dry Blotting System, some of my proteins blew through the membrane. What could have caused this?

This could happen if the transfer time used is too long. We recommend reducing the transfer time by 30 second increments.
Note: Pre-stained markers are charged, so tend to blow-through more than regular proteins.

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

I transferred my proteins using the iBlot Dry Blotting System but the high-molecular weight proteins remained in the gel indicated by staining of the gel after transfer. Can you help me troubleshoot?

This could happen if an incorrect voltage Method was used or if inappropriate transfer conditions were used. Make sure that the voltage Method and run time used is correct, based on the gel type, as described on page 13 in the manual.

For mini or midi gels:
- Perform an ethanol equilibration step as described on page 28 in the manual to improve transfer.
- Use a lower gel percentage to separate the high-molecular weight proteins.
- Increase the transfer time in 30-second increments.

For E-PAGE gels:
- Increase the transfer time in 30-second increments.
- Use Method P2 for 8 minutes.

Note: It is normal for some proteins to remain in the gel because some high-molecular weight proteins do not transfer completely using the iBlot Gel Transfer Device, compared to semi-wet transfer apparatus.

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

I see a lot of empty spots on the membrane after transfer using the iBlot Dry Blotting System. What could have caused this?

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 using the De-bubbling Roller for E-PAGE gels or Blotting Roller for other gels.
- Expired or creased membranes used. Use the iBlot Gel Transfer Stacks before the expiration date printed on the package.

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

I used the iBlot Dry Blotting System and none of my proteins transferred to the membrane. What could have happened?

This could be due to no current passing through or because an incorrect voltage Method was used. Make sure that the electrical circuit is complete and current is flowing through the device. Please check to make sure that the correct voltage Method is used, see page 13 in the manual (http://tools.thermofisher.com/content/sfs/manuals/iblotsystem_man.pdf).

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

With the iBlot Dry Blotting System, the iBlot Anode Stack, Bottom transfer gel melts to a viscous blue solution. How can I prevent this from happening again?

This happens if the membrane is trimmed to fit the gel size resulting in direct contact between the iBlot Cathode, Top and Anode Bottom stacks. Always maintain the membrane size identical to the transfer stack. Transfer quality is not affected by smaller gel size compared to the membrane.

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

Sometimes there is green discoloration on the membrane edges around the gel when using the iBlot Dry Blotting System. What is this, and does it affect the results?

The green discoloration is due to copper ions from the iBlot Transfer Stacks being carried by liquids and deposited on the membrane. These deposits do not interfere with downstream processes. The stained regions can be cut away, but membrane washing typically results in their removal. To minimize this effect, shake excess water off the filter paper and buffer from the gel before placing each on the stack.

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

After transferring my gel using the iBlot Dry Blotting System, both the membrane and the gel turned blue. Why is this?

Longer transfer times result in the deposition of copper ions, causing the blue discoloration. Be sure to perform the transfer for the recommended time for each gel type.

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

I use the iBlot Dry Blotting System and see some corrosion of the iBlot Cathode Stack, Top. What is causing this?

This is most likely due to incorrect placement of the top Stack. Be sure the iBlot Cathode Stack, Top is placed correctly with the copper electrode side facing up. Avoid placing the top stack in the inverted position.

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

I am getting an "Error2" message displayed with the iBlot Dry Blotting System. What is the problem?

Here are possible causes and solutions:

- The iBlot Cathode Stack, Top is touching the copper electrode on the iBlot Anode Stack, Bottom: Open the lid and align the iBlot Cathode Stack, Top to the right. Continue the run by briefly pressing Start/Stop Button or restart the run by pressing and holding the Start/Stop Button.
- The layers are not aligned: Align the layers properly as described in the protocols. Ensure that the electrodes are in contact.
- Current is above 5.5 amp: Select a program with a lower voltage. Open the iBlot Lid and ensure the stacks are aligned properly. Close the lid and restart the run by subtracting the time already elapsed. Replace the iBlot Gel Transfer Stacks with fresh transfer stacks. Ensure an iBlot Filter Paper was used during blotting of mini- or midi-gels.

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

I am getting an "Error1" message displayed with the iBlot Dry Blotting System. What is wrong?

This indicates an open electrical circuit during the run. It occurs if the lid opened during the run.
Close the lid and continue the run by briefly pressing the Start/Stop Button or restart the run by pressing and holding the Start/Stop Button.

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

I am using the iBlot Dry Blotting System and no current seems to be passing through (red light is not on after securing the lid). Can you help me troubleshoot?

This indicates an incomplete electric circuit and here are possible causes and solutions:

- iBlot Disposable Sponge covers the metal contact or the metal contact on the sponge is on the left: Reinsert the iBlot Disposable Sponge such that the metal contact on the sponge is on the top right of the lid and is in contact with the electrode on the transfer stack.
- Incorrect position of the transfer stacks or improper assembly of the transfer stacks: Make sure the transfer stack is placed in the proper position in the blotting surface to allow proper contacts with the electrodes. Ensure the transfer stacks are assembled correctly; use the iBlot Anode Stack, Bottom first followed by the gel and iBlot Cathode Stack, Top.
- Incorrect position of the pull tab: Ensure the pull tab from the iBlot Cathode Stack, Top is towards the right of the assembly in the blotting surface.
- iBlot Anode Stack, Bottom placed on the device without the tray including the electrical contact: Do not remove the iBlot Anode Stack, Bottom from the tray during the assembly. The blotting is performed with the bottom stack in the plastic tray.
- iBlot Cathode Stack, Top placed on the device with the tray: Always remove the iBlot Cathode Stack, Top from the red plastic tray before placing the top stack on the assembly. Do not use the iBlot Cathode Stack, Top with the tray.
- The metal safety contacts in the lid hinge may be dirty and do not make contact: Clean the metal safety contacts in the lid hinge with a cotton swab and water.

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

Are the iBlot Transfer Stacks compatible with Li-COR detection?

Yes, the iBlot Transfer Stacks are compatible with Li-COR detection.

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

What is the Blotting Roller that is provided with the iBlot Dry Blotting System?

The Blotting Roller is a plastic roller attached to a stainless steel handle. It is used to remove any air bubbles between the gel and blotting membrane during the assembly of the stacks and gel.

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

What is the De-bubbling Roller?

The De-bubbling Roller is a stainless steel, aluminum roller designed to remove any air bubbles between the gel and blotting membrane during the assembly of the stacks when blotting E-PAGE gels. We recommend using the De-bubbling Roller only for E-PAGE gels. Other gel types may stretch and tear if pulled through the roller. The De-bubbling Roller is provided with the iBlot Dry Blotting System but not with the iBlot 2 Dry Blotting System.

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

What is the iBlot E-PAGE tab?

The iBlot E-PAGE Tab is a steel tab used during blotting of E-PAGE gels. It is attached to the iBlot Cathode Stack, Top and is used to pull the transfer stack assembly towards the blotting surface during the de-bubbling process of E-PAGE gels.

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

What is the purpose of the iBlot Filter Paper?

The iBlot Filter Paper is used for blotting mini- or midi-gels. It is placed on top of the gel before placing the iBlot Cathode Stack (Top Stack) to protect the gel integrity during the blotting process. The iBlot Filter Paper is supplied in two sizes for efficient blotting of mini- and midi-gels. We do not recommend using the iBlot Filter Paper for blotting E-PAGE gels.

Note: Failure to use the iBlot Filter Paper during blotting of mini- or midi-gels may result in high currents exceeding the current limit leading to an error (Error2) during the run.

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

Is there a stripping protocol for the iBlot Dry Blotting System?

A conventional stripping protocol using 0.1 M glycine, pH 2, works with polyclonal antibodies.

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

How can I get better transfer of high-molecular weight proteins with the iBlot Dry Blotting System?

Proteins larger than ~150 kDa migrate more slowly than smaller proteins. Therefore, more time is required to transfer them from the gel to the membrane. We recommend using P3 program with a transfer time of 8-10 minutes for optimal transfer of these proteins.

To enhance transfer efficiency, we also recommend adding an equilibration (gel-soaking) step between electrophoresis and western transfer and using NuPAGE Invitrogen 3-8% Tris-acetate gels for electrophoresis. For the protocol, please see Transferring Large and Small Proteins Using the iBlot Dry Blotting System Application Note (http://www.thermofisher.com/content/dam/LifeTech/migration/en/filelibrary/protein-expression/pdfs.par.87290.file.dat/iblot%20small%20and%20large%20protein%20app%20note.pdf).

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

Are the copper electrodes in the transfer stacks of the iBlot Dry Blotting System recyclable?

The electrodes used in our iBlot Transfer Stacks are copper-coated nylon, and the amount of copper left in the electrodes after a transfer is rather small. There is 0.6 grams of copper per sheet before transfer, and even less after transfer. To maximize the recovery of the copper mesh electrodes, we have established an agreement with a recycling center in the United States. In order to prepare the copper electrodes for recycling, please follow the instructions listed here (https://www.thermofisher.com/content/dam/LifeTech/migration/files/proteins-expression-isolation-analysis/pdfs.par.0358.file.dat/iBlot%20copper%20reycling%20instructions%20NA%20only.pdf).

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

I want to perform western transfer with mini gels (8 x 8 cm), but I do not have iBlot Transfer Stacks (Mini). Can I use iBlot Transfer Stacks (Regular) to transfer my mini gels?

It is best not to transfer a single mini gel on a regular-sized transfer stack. Although in most cases the transfer will be fine, empty spaces on the transfer stack that are not in direct contact with a gel could potentially cause distortions across the whole surface of the membrane, including the portion in contact with the gel. It is best to have more than 50% of a membrane in contact with the gel, if possible.

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

Is it okay to freeze the iBlot Gel Transfer Stacks?

The iBlot Gel Transfer stacks should not be frozen as they contain a gel matrix that will be damaged by freezing.

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

Do you offer replacement lid latches and electrodes for the iBlot Gel Transfer Device?

Yes, the Cat. No. for the iBlot lid latch is IB1003 and the Cat. No. for the iBlot Electrodes (1 pair) is IB1002. Here are the instructions to replace the electrodes (http://tools.thermofisher.com/content/sfs/manuals/iblot_electrode_replacement_man.pdf).

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

Is the PVDF membrane in the iBlot PVDF Gel Transfer Stack pre-activated?

The PVDF membrane is pre-activated and ready for use without any pretreatment with alcohol.

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

What is the recommended storage condition for the iBlot Transfer Stacks?

We recommend storing the iBlot Transfer Stacks at room temperature.

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

What are the different kinds of iBlot Transfer Stacks that you offer and are they available separately?

The following iBlot Transfer Stacks are available for use with the iBlot Gel Transfer Device and are also sold separately:

- iBlot Gel Transfer Stacks are used to transfer proteins from gels onto nitrocellulose or PVDF membranes (western blotting). Both nitrocellulose and PVDF transfer stacks are available in mini and regular formats.
- iBlot DNA Transfer Stacks are used to transfer DNA from gels onto nylon membranes (Southern blotting).

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

What kind of gels can I use with the iBlot Dry Blotting System for western blotting?

The iBlot Dry Blotting System is compatible for use with Bolt Bis-Tris Plus, NuPAGE Bis-Tris, Tris-Acetate, Tris-Glycine, Tricine (in mini- and midi-gel formats), and E-PAGE gels.

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

Do you still offer the iBlot Gel Transfer Device?

The iBlot Gel Transfer Device has been discontinued and we have launched the iBlot 2 Gel Transfer Device, that is an improved version. The iBlot Transfer Stacks, Western Detection Stacks and DNA Transfer Stacks are still available for purchase and are to be used exclusively with the original iBlot Gel Transfer Device, and are not compatible with the new iBlot 2 Gel Transfer Device (http://www.thermofisher.com/order/catalog/product/IB21001). Only iBlot 2 consumables are to be used with the iBlot 2 Gel Transfer Device.

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

Does the iBlot Dry Blotting System work with native or native-blue gels?

Yes, please take a look at the Western Blotting NativePAGE Invitrogen Bis-Tris Gels Using the iBlot 7-Minute Blotting System Application Note (http://www.thermofisher.com/content/dam/LifeTech/migration/en/filelibrary/pdf.par.18870.file.dat/native-with-iblot-app-note-v3.pdf).

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