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View additional product information for iBlot™ Transfer Stack, nitrocellulose, mini - FAQs (IB301002, IB301032)
48 product FAQs found
大多数研究人员使用硝化纤维素或PVDF膜以用于SuperSignal West Pico化学发光底物。两种效果都很好，但是硝化纤维素膜在一些应用中比PVDF膜更合适。此外，对于这种底物，荷电尼龙膜效果较好。
大多数研究人员使用硝化纤维素或PVDF膜以用于SuperSignal West Femto化学发光底物。两种效果都很好，但是硝化纤维素膜在一些应用中比PVDF膜更合适。此外，对于这种底物，荷电尼龙膜效果较好。
大多数研究人员使用硝化纤维素或PVDF膜以用于SuperSignal West Dura化学发光底物。两种效果都很好，但是硝化纤维素膜在一些应用中比PVDF膜更合适。此外，对于这种底物，荷电尼龙膜效果较好。
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。
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。
兼容，iBlot转印膜组可以兼容Li-COR检测。
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（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）。
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。
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。
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）。
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凝胶转印膜块不可以冷冻，因为冷冻会破坏其中含有的凝胶基质。
我们建议将iBlot转印膜块保存于室温。
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）。
Most researchers use nitrocellulose or polyvinyldiflouride (PVDF) membranes with SuperSignal West Dura Chemiluminescent Substrate. Both work well, although nitrocellulose seems to be better suited in some applications than PVDF. In addition, charge-modified nylon membrane performs well with this substrate. Please also see our guide for choosing western blot membranes (https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-assays-analysis/western-blotting/transfer-proteins-western-blot/membranes-transfer-buffers-western-blotting/membranes-western-blotting.html).
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
Most researchers use nitrocellulose or polyvinyldiflouride (PVDF) membranes with SuperSignal West Femto Maximum Sensitivity Substrate. Both work well, although nitrocellulose seems to be better suited in some applications than PVDF. In addition, charge-modified nylon membrane performs well with this substrate. Please also see our guide for choosing western blot membranes (https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-assays-analysis/western-blotting/transfer-proteins-western-blot/membranes-transfer-buffers-western-blotting/membranes-western-blotting.html).
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
Most researchers use nitrocellulose or polyvinyldiflouride (PVDF) membranes with SuperSignal West Dura Extended Duration Substrate. Both work well, although nitrocellulose seems to be better suited in some applications than PVDF. In addition, charge-modified nylon membrane performs well with this substrate. Please also see our guide for choosing western blot membranes (https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-assays-analysis/western-blotting/transfer-proteins-western-blot/membranes-transfer-buffers-western-blotting/membranes-western-blotting.html).
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.