iBlot™ 2 Gel Transfer Device - FAQs

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101 product FAQs found

我使用iBlot 2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 2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 2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 2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 2干转系统转印蛋白质时，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Blotting Roller将凝胶压平。为得到良好的效果，请遵循使用手册（https://tools.thermofisher.com/content/sfs/manuals/iblot2_device_man.pdf）第19页的建议。

使用iBlot 2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 2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/iblot2_device_man.pdf）第17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：
•按照使用手册第35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 2凝胶转印设备对某些高分子量蛋白质的转印不完全。

使用iBlot2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 2凝胶转印膜组。

我在使用iBlot 2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/iblot2_device_man.pdf）第17页）。

使用iBlot2干转系统转印后，我发现底部模组转印凝胶熔化成了粘稠的蓝色液体。这是为什么？

若将膜修剪成符合凝胶大小的尺寸，会导致顶部和底部膜组直接接触，从而发生熔化情况。保持膜的尺寸与转印膜组一致，可避免这种情况的发生。与膜相比，较小的凝胶尺寸不会影响转印质量。

使用iBlot 2干转系统时，膜的边缘有一点变绿。这是否会影响结果？

变为绿色是由于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。

使用iBlot 2干转系统进行凝胶转印后，膜和凝胶都变成蓝色了。这种情况正常吗？

转印时间过长可导致铜离子沉积。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 2干转系统时，发现顶部膜组被腐蚀。可能原因是什么？

这可能是因为顶部膜组的位置不正确。应确保正确放置顶部膜组，使铜电极一侧朝上。不要将顶部膜组反向放置。

我在使用iBlot 2干转系统时，无电流通过。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 2吸收垫接头的位置不正确：应确保iBlot 2吸收垫的电极接头对准iBlot2凝胶转印设备转印表面上对应的电极接头。
- 放在设备上的顶部膜组方向颠倒：应确保顶部膜组的铜电极朝上。
- 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。
- 安装膜组时，未去除塑料分隔器： 应确保取出膜组中的塑料分隔器[隔片]（见使用手册（https://tools.thermofisher.com/content/sfs/manuals/iblot2_device_man.pdf）第21页）。

我正在以恒定电压进行Tris-甘氨酸凝胶转印，但电流读数高于预期起始电流。可能因为什么？

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。

我将蛋白质置于Tris-甘氨酸凝胶上在非变性条件下进行电泳。蛋白质的pI高于Tris-甘氨酸转膜缓冲液的pH。你们建议如何对蛋白质进行转印？

•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。

我在对NuPAGE凝胶上的大分子蛋白质进行转印时遇到问题。你们有何建议？

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。

转印后，为什么膜上出现空白的点？

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。

我在蛋白质转印后发现膜上的条带呈扩散状和旋涡状。可能原因是什么？

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。

我在进行蛋白质转印时，电源在转印中途关闭。哪里出错了？

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。

在蛋白质转印后对凝胶进行染色，发现有大量蛋白质仍留在凝胶中。我该怎么办？

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。

在蛋白质转印后，我发现有大量蛋白质穿过了膜，因为第二张膜上也有蛋白质。你们能否提供帮助？

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。

我进行蛋白质转印后，发现没有蛋白质转印到膜上。你们有何建议？

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。

以恒定电压条件进行蛋白质转印时，什么原因会导致实际电流远远超过预期起始电流？

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。

我正在以恒定电压进行凝胶转印，但电流读数比预期起始电流低很多。可能原因什么？

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。

iBlot 2吸收垫是什么？

iBlot 2吸收垫可吸收转印过程中膜块上形成的多余液体，并对膜块整体施加均匀的压力。在转印前将吸收垫置于组装好的iBlot 2膜块上方。我们建议iBlot 2吸收垫使用一次即丢弃。

iBlot 2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，可用于除去在安装膜块和凝胶期间凝胶与印迹膜之间的所有气泡。

我能否将iBlot 2转印膜块的膜剪切成为适合我的分离凝胶的尺寸？

不能。保持膜的尺寸与转印膜块一致。这有助于确保顶部和底部膜块之间无直接接触。

iBlot 2 PVDF和硝化纤维素膜能否用于荧光抗体或膜染色（如LI-COR、丽春红S）？

iBlot 2 PVDF和硝化纤维素膜可用于所有常用的检测方法，如染色、免疫检测、荧光检测等。

能否使用我自选的膜替代iBlot 2干转系统中的膜？

可以，但是结果可能不理想。

使用iBlot 2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8-10分钟来转印这类蛋白，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 3-8% Tris-acetate凝胶进行电泳。请参考使用手册第35页的实验方案。

iBlot 2干转系统能否用于转印小分子量和大分子量的蛋白质？

可以。但是，转印某些蛋白质时，可能需要稍微对转印方案（比如步骤、电压、时间等）进行优化。

iBlot 2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 2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。

我想对小型凝胶（8 x 8 cm）进行蛋白质转印，但我没有iBlot 2转印膜块（小型）。能否使用iBlot 2转印膜块（标准）进行小型凝胶转印？

最好不要在标准规格的转印膜块中单独转印一块小型凝胶。尽管在大多数情况下能够实现良好的转印，但转印膜块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，最好使至少50%的膜与凝胶接触。因此，建议使用标准规格的转印膜块同时转印2块小型凝胶。

与iBlot凝胶转印膜块不同，iBlot 2顶部膜块中的转印凝胶层下方有一层白色的薄片，这有什么作用？

这是一个塑料分隔器，用于分隔顶部膜块和底部膜块。应在安装膜块之前，从顶部膜块中取出分隔器并丢弃。

注意：在某些情况下，膜可能会粘到分隔器上。如果出现这样的情况，应该用镊子取下膜并将膜放到底部膜块上方。

你们是否提供iBlot 2凝胶转印设备的替换用电极？

是的，我们提供iBlot 2电极替换套装，货号IB28001，包含2个电触头、2个螺丝和1个圆形缓冲垫。请参考使用手册第37页的更换说明。

iBlot 2凝胶转印膜块能否冷冻？

iBlot 2凝胶转印膜块不可冷冻，因为冷冻会破坏其中含有的凝胶基质。

能否使用超过有效期的iBlot 2转印膜组？

不能。使用超过有效期的iBlot 2转印膜块可能会得到不理想的转印结果。

iBlot 2转印膜组的质保期是多久？

一般为9个月。有效期印于包装盒上。

iBlot 2转印膜块的推荐保存条件使什么？

我们建议将iBlot 2转印膜块保存于室温。

iBlot 2转印膜组有哪些不同种类？能否单独购买？

我们可提供iBlot 2凝胶转印膜块和完整的硝化纤维素膜或PVDF膜，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）。这些膜块均可单独购买。

进行免疫印迹分析时，哪类凝胶可用于iBlot 2干转系统？

iBlot 2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（仅E-PAGE 48）凝胶。

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）。

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滤纸能否用于E-PAGE凝胶转印？

我们不建议将iBlot滤纸用于E-PAGE凝胶转印。

你们推荐使用哪种方法进行E-PAGE凝胶转印？

建议使用iBlot/iBlot 2干转系统对E-PAGE凝胶进行转印（见E-PAGE技术指南（https://tools.thermofisher.com/content/sfs/manuals/epagetechguide_man.pdf）第27页）。请注意，该系统仅适用于E-PAGE 48凝胶。也可采用Invitrogen半干转仪（货号SD1000，第33页）进行半干转或采用半湿转法（第37页）。

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）。

If a green color appears on the membrane after the transfer using the iBlot 2 Dry Blotting System, will it affect the results?

The green discoloration is copper deposits from the transfer stack, 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.

Can I cut the bottom stack of my iBlot 2 Transfer Stack to fit my separating gel?

No. Do not trim the iBlot 2 Transfer Stacks to fit your gel size.

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

Is the PVDF membrane in the iBlot 2 PVDF Gel Transfer Stack pre-activated?

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

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

Can I use the stack and/or absorbent pad more than once when using the iBlot 2 Dry Blotting System?

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.

Can I perform multiple transfers, with a new transfer starting immediately after the previous one when using the iBlot 2 Dry Blotting System?

Yes. The device is designed to do so with no impact on performance.

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

Can I create my own transfer conditions with the iBlot 2 Dry Blotting System?

Yes. The device allows programming custom methods.

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

What types of membranes are available in the iBlot Transfer Stacks?

iBlot Transfer Stacks are available with either an integrated 0.2 µm nitrocellulose membrane or a 0.2 µm PVDF membrane. Both types are available in two sizes: a mini size which can accommodate one mini gel (8 x 8 cm), or regular size which can accommodate one E-PAGE gel, one midi gel (8 x 13 cm), or two mini gels (8 x 8 cm).

The catalog numbers are as follows:
IB3010-01 iBlot Gel Transfer Stacks, Regular (10 Blots)
IB3010-02 iBlot Gel Transfer Stacks, Mini (10 Blots)
IB4010-01 iBlot Transfer Stack PVDF, Regular (10 Blots)
IB4010-02 iBlot Transfer Stack PVDF, Mini (10 Blots)

If you would like to blot proteins onto your own membrane rather than the supplied nitrocellulose or PVDF, you can replace the integrated membrane with your desired membrane as described below:

1) Wet the desired blotting membrane in deionized water. (For PVDF membranes, first wet the PVDF membrane in 100% methanol and then rinse in deionized water).
2) Carefully remove the nitrocellulose membrane from the bottom stack using forceps.
3) Place the wetted blotting membrane on the bottom transfer stack. (Membrane should be completely wet, but not dripping; make sure there is not too much excess water.)
4) Be sure to align the membrane flush to the bottom stack gel, and remove any air bubbles using the blotting roller. Proceed with the standard iBlot transfer protocol.

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

Are the iBlot 3 Transfer Stacks compatible with the original iBlot and iBlot 2 transfer devices?

Unfortunately, the iBlot 3 Transfer Stacks are not compatible with the original iBlot and iBlot 2 transfer devices. The iBlot 3 Transfer Stacks are only compatible with the iBlot 3 Western Blot Transfer Device.

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

How many gels can I transfer simultaneously using iBlot 2 Transfer Stacks?

You can transfer:
- One midi gel or two mini gels (head-to-head) using the iBlot 2 Regular Transfer Stacks
- One mini gel using iBlot 2 Mini Transfer Stacks

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

I used the iBlot 2 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 2 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 2 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 2 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 using the iBlot 2 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 gel is properly flattened by using the Blotting Roller. Follow the recommendations on page 19 in the manual (http://tools.thermofisher.com/content/sfs/manuals/iblot2_device_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 2 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 2 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 17 in the manual (http://tools.thermofisher.com/content/sfs/manuals/iblot2_device_man.pdf).

For mini or midi gels:
- Perform an ethanol equilibration step as described on page 35 in the manual (http://tools.thermofisher.com/content/sfs/manuals/iblot2_device_man.pdf) 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 P3 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 2 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 2 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 using the Blotting Roller.
- Expired or creased membranes used. Use the iBlot 2 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 2 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 17 in the manual (http://tools.thermofisher.com/content/sfs/manuals/iblot2_device_man.pdf).

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

After transferring using the iBlot 2 Dry Blotting System, I noticed that the Bottom Stack transfer gel melted to a viscous blue solution. Why is this?

This happens if the membrane is trimmed to fit the gel size resulting in direct contact between the Top and Bottom stacks. This can be avoided by maintaining 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.

I used the iBlot 2 Dry Blotting System and noticed some green discoloration of the edges of the membrane. Will this affect the results?

The green discoloration is due to copper ions carried with liquids that get deposited onto 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.

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

After transferring using the iBlot 2 Dry Blotting System, both my membrane and the gel turned blue. Is this normal?

Longer transfer times result in the deposition of copper ions. 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 used the iBlot 2 Dry Blotting System and noticed that the Top Stack has been corroded. What could have caused this?

This could be caused by incorrect placement of the Top Stack. Please check to make sure that the Top Stack is placed correctly with the copper electrode 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 using the iBlot 2 Dry Blotting System and no current seems to be passing through. Can you help me troubleshoot?

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

- Incorrect placement of iBlot 2 Absorbent Pad contact: Make sure the electrical contact of the iBlot 2 Absorbent Pad is aligned with the corresponding electrical contacts on the blotting surface of the iBlot2 Gel Transfer Device.
- Top Stack placed on the device upside down: Make sure the Top Stack is assembled with the copper electrode facing up.
- 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.
- Plastic separator was not removed when assembling stack: Make sure that the plastic separator is removed from the stack, see page 21 in the manual (http://tools.thermofisher.com/content/sfs/manuals/iblot2_device_man.pdf).

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

I am transferring a Tris-Glycine gel using constant voltage and the current reading is way over the expected starting current. Can you offer some suggestions?

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.

I ran my protein under native conditions on a Tris-Glycine gel. It has a pI that is higher than the pH of the Tris-Glycine transfer buffer. Can you offer some tips for transferring it?

- 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.

I had problems transferring my larger-molecular weight proteins from my NuPAGE gel. Can you please offer some suggestions?

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.

What causes empty spots on my membrane after transfer?

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.

I performed a western transfer and see the appearance of diffuse bands and swirling patterns on the membrane. What could have happened?

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.

When I perform a western transfer, the power supply shuts off in the middle of the transfer. What is wrong?

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.

After a western transfer, I noticed that a significant amount of protein remained in the gel indicated by staining of the gel after transfer. What should I do?

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.

After a western transfer, I noticed that a significant amount of protein had passed through the membrane indicated by the presence of proteins on the second membrane. Can you please help?

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.

I performed a western transfer and none of my proteins transferred to the membrane. Can you offer some tips?

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.

During western transfer conditions using constant voltage, what would cause the actual current to greatly exceed the expected starting current?

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.

I am transferring my gel using constant voltage and the current reading has dropped much lower than the expected starting current. What could have happened?

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.

What is the iBlot 2 Absorbent Pad?

The iBlot 2 Absorbent Pad absorbs any excess liquid formed on the stacks during blotting and generates even pressure on the stack assembly. It is placed on top of the assembled iBlot 2 stack prior to transfer. We recommend discarding the iBlot 2 Absorbent Pad after every use.

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 2 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.

Can I cut the membrane in the iBlot 2 Transfer Stacks to fit my separating gel?

No. Maintain the membrane size identical to the transfer stacks. This helps ensure that there is no direct contact between the top and bottom transfer stacks.

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

Are the iBlot 2 PVDF and nitrocellulose membranes compatible with fluorescent antibodies or membrane staining (e.g., LI-COR, Ponceau S)?

The membranes are compatible with all commonly used detection methods such as staining, immunodetection, fluorescence, etc.

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

Can I replace the membrane with my preferred membrane when using the iBlot 2 Dry Blotting System?

It is possible but the results may be inferior.

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 using the iBlot 2 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 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 3-8% Tris-acetate gels for electrophoresis. For the protocol, please refer to page 35 in the manual (http://tools.thermofisher.com/content/sfs/manuals/iblot2_device_man.pdf).

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

Can the iBlot 2 Dry Blotting System be used for transfer of low- and high-molecular weight proteins?

Yes. However, minor optimizations in the transfer protocol (i.e., steps, voltage, time) may be needed for some proteins.

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 2 Dry Blotting System recyclable?

To maximize the recovery of the copper mesh electrodes used in our iBlot 2 transfer stacks, 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%202%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 2 Transfer Stacks (Mini). Can I use iBlot 2 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. So the recommendation would be to transfer 2 mini gels using a Regular stack.

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

Unlike the iBlot Gel Transfer Stacks, in the iBlot 2 Top Stack, I see a white sheet beneath the Transfer gel layer. What purpose does it serve?

This is the plastic separator that separates the Top Stack from the Bottom Stack. Right before assembling the stack, this separator needs to be removed from the Top Stack and discarded.

Note: In some instances, the membrane may adhere to the separator. If this is the case, use forceps to remove the membrane and place it on top of the Bottom Stack.

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

Do you offer replacement electrodes for the iBlot 2 Gel Transfer Device?

Yes, we offer the iBlot 2 Electrode Replacement Kit, Cat. No. IB28001, that consists of two electrical contacts, two screws, and a round bumper. Please refer to page 37 in the manual (http://tools.thermofisher.com/content/sfs/manuals/iblot2_device_man.pdf) for the instructions.

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

Is it okay to freeze the iBlot 2 Gel Transfer Stacks?

The iBlot 2 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.

Can I use the iBlot 2 Transfer Stacks after the expiration date?

No. The results may be inferior after the expiration date.

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

What is the shelf life of iBlot 2 Gel Transfer Stacks?

It is currently 9 months, and the expiration date is printed on the box.

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 2 Transfer Stacks?

We recommend storing the iBlot 2 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 2 Transfer Stacks that you offer and are they available separately?

We offer iBlot 2 Gel Transfer Stacks with integrated nitrocellulose or PVDF membranes for transferring proteins from gels onto nitrocellulose or PVDF membranes, respectively (western blotting). These stacks can also be purchased separately.

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 2 Dry Blotting System for western blotting?

The iBlot 2 Dry Blotting System is compatible for use with Bolt Bis-Tris, NuPAGE Bis-Tris, Tris-Acetate, Tris-Glycine, Tricine (in mini- and midi-gel formats), and E-PAGE gels (E-PAGE 48 only).

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.

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.

Can I use the iBlot Filter Paper for blotting E-PAGE gels?

We do not recommend using the iBlot Filter Paper for blotting E-PAGE gels.

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

What method do you recommend for blotting E-PAGE gels?

The preferred method of transfer for E-PAGE gels is dry blotting using the iBlot/iBlot 2 Dry Blotting System (see Page 27 of the E-PAGE Technical Guide http://tools.thermofisher.com/content/sfs/manuals/epagetechguide_man.pdf). Please note that this works only for E-PAGE 48 gels. Semi-dry blotting using the Invitrogen Semi-Dry Blotter, Cat. No. SD1000 (Page 33), or semi-wet blotting (Page 37) may also be performed.

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.