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View additional product information for iBlot™ 2 Transfer Stacks, nitrocellulose, mini - FAQs (IB23002)
66 product FAQs found
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。
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。
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。
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。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。
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。
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）。
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。
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。
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。
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。
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）。
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。
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。
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和硝化纤维素膜可用于所有常用的检测方法，如染色、免疫检测、荧光检测等。
可以，但是结果可能不理想。
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。
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。
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。
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。
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。
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。
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。
一般为9个月。有效期印于包装盒上。
我们建议将iBlot 2转印膜块保存于室温。
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。
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）。
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.
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.
The PVDF membrane is pre-activated and ready for use without any pre-treatment with alcohol.
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No. The transfer stacks have a finite amount of ions to drive the transfer and are depleted after a single use.
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Yes. The device is designed to do so with no impact on performance.
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Yes. The device allows programming custom methods.
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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.
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.
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.
This is likely due to use of TBST buffers for washing. We recommend using PBST or WesternBreeze wash solutions.
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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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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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.
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The membranes are compatible with all commonly used detection methods such as staining, immunodetection, fluorescence, etc.
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It is possible but the results may be inferior.
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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.
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.
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.
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.
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.
The iBlot 2 Gel Transfer stacks should not be frozen as they contain a gel matrix that will be damaged by freezing.
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No. The results may be inferior after the expiration date.
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It is currently 9 months, and the expiration date is printed on the box.
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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.
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.
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.
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.