E-PAGE™ Midi Protein Gels, 8%, 3.7 mm, 48-well, 8 gels (1 kit) - FAQs

在Beckman Coulter's Biomek FX工作站上使用E-Gel 96琼脂糖凝胶和E-PAGE 96凝胶进行电泳的自动化脚本可在我们的网站上找到：www.thermofisher.com/egels（在左侧导航窗格内点击Labware Definitions链接）。

EG程序用于E-Gel 96和48凝胶电泳，而EP用于E-PAGE 96和48凝胶电泳。

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。

对于E-PAGE 48凝胶的半干转，我们建议使用含15%甲醇的2X NuPAGE转膜缓冲液；对于E-PAGE 96凝胶的半干转，我们建议使用无甲醇的2X NuPAGE转膜缓冲液。在25V恒定电压条件下转印30-60分钟。请参考E-PAGE技术指南（https://tools.thermofisher.com/content/sfs/manuals/epagetechguide_man.pdf）第33页。

E-PAGE凝胶转印的推荐方法是使用iBlot/iBlot 2干转系统进行干转（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）。

我们不建议将iBlot滤纸用于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）。

我们建议保持Mother E-Base设备和Daughter E-Base设备表面无污染物。清洁设备时，先将基座与电源断开，再用干布擦拭。不要试图打开或维修基座。

E-Holder平台专为在上样期间支持E-PAGE凝胶而设计。在对多块凝胶上样而其他凝胶正在E-Base设备上电泳时，您可选择使用E-Holder平台。

注意：E-Holder平台不是电源装置，不可连接到插座，不可用于E-PAGE 48或96凝胶电泳。为了获得最佳结果，应在使用E-Holder平台上样后15分钟内，使E-PAGE凝胶在Mother E-Base设备或Daughter E-Base设备上开始电泳。

我们推荐使用EP程序进行E-PAGE凝胶电泳。

E-PAGE凝胶可兼容多种标准Coomassie染色、银染或荧光染色方案。E-PAGE凝胶比大部分SDS-PAGE小型凝胶更厚，所以染色和脱色步骤需要更长时间。我们推荐使用以下染料进行E-PAGE凝胶染色。E-PAGE技术指南（https://tools.thermofisher.com/content/sfs/manuals/epagetechguide_man.pdf）第40-49页对染色方案进行了详细描述。

总蛋白质染料：
•SYPRO Ruby蛋白质凝胶染料（第40页）
•Coomassie染料（第42页）
•SimplyBlue SafeStain（第44页）
•SilverQuest银染试剂盒（第47页）
•SilverXpress银染试剂盒（第48页）

专用蛋白质染料：
•Lumio绿色检测试剂，用于检测Lumio融合蛋白（第40页）
•InVision His-tag In-Gel Stain，用于检测6X His标签蛋白（首先推荐转印，第49页）

电泳结束后，可使用Butterfly Opener（与E-PAGE凝胶一起提供）打开E-PAGE凝胶盒，从而将凝胶用于免疫印迹分析或染色等下游应用。

电泳：
对E-PAGE 48 8%凝胶，推荐使用SeeBlue Plus 2预染标准品，货号LC5925
对E-PAGE 96 6%凝胶，推荐使用E-PAGE SeeBlue预染标准品，货号LC5700

免疫印迹分析：
对E-PAGE 48 8%凝胶，推荐使用 MagicMark XP Western蛋白质标准品，货号LC5602
对 E-PAGE 96 6%凝胶，推荐使用 E-PAGE MagicMark未染色蛋白质标准品，货号LC5701

荧光检测：
对 E-PAGE 48 8%凝胶，推荐使用BenchMark荧光蛋白标准品，货号LC5928
对E-PAGE 96 6%凝胶，推荐使用BenchMark荧光蛋白标准品，货号LC5928

E-PAGE 96 6%和E-PAGE 48 8%凝胶的推荐电泳时间分别为14分钟和25分钟。无需预电泳。可以通过延长电泳时间来改善分辨率，但应注意不要使电泳进入下一个孔。E-PAGE 96或48凝胶的电泳时间分别不应超过25和30分钟。电泳功率为9.5瓦特。

我们推荐使用大型凝胶干燥试剂盒（货号NI2207）进行E-PAGE凝胶干燥。也可以风干E-PAGE凝胶。E-PAGE 96凝胶至少需要4天才能完全风干。不建议使用真空干燥——这类凝胶的厚度使其非常容易破裂。

请遵循E-PAGE技术指南（https://tools.thermofisher.com/content/sfs/manuals/epagetechguide_man.pdf）第10页中的建议。高盐或高洗涤剂含量的样品可使E-PAGE凝胶的分辨率降低。

4X E-PAGE上样缓冲液1的成分是保密的。

我们推荐使用Lumio绿色检测试剂盒中提供的Lumio凝胶样品缓冲液进行胶内检测。不建议使用E-PAGE上样缓冲液。Lumio融合蛋白显色时，无需从凝胶盒中取出E-PAGE凝胶。

我们推荐使用4X E-PAGE上样缓冲液1（与E-PAGE凝胶一起提供）制备用于SDS-PAGE电泳和凝胶染色或转印的样品。我们不推荐使用任何其他SDS样品缓冲液，如NuPAGE LDS样品缓冲液和Tris-甘氨酸SDS样品缓冲液，否则可能导致条带分辨率降低。

E-PAGE 48和E-PAGE 96凝胶的推荐总上样体积为15µL（最多20µL）。上样体积太小会导致分辨率较差和拖尾效应。为实现较好的条带分离，我们推荐保持样品间体积均一，并在空孔中加入去离子水。

E-PAGE凝胶每个泳道的推荐上样量是1–20 μg蛋白质。每个泳道的最大推荐蛋白质上样量是20 μg。过多的蛋白质会导致分辨率较差和拖尾效应。

E-PAGE凝胶含有SDS，推荐将其用于变性条件下的电泳。

E-PAGE 96 6%和E-PAGE 48 8%凝胶上每条泳道的电泳距离分别为1.6 cm和3.2 cm。

E-PAGE 96 6%凝胶和E-PAGE 48 8%凝胶可分离的蛋白质分子量范围分别是10–220 kDa和10–200 kDa。

E-PAGE凝胶不含防腐剂。它们在生产过程中经过紫外消毒。

E-PAGE凝胶的配方是保密的。

E-PAGE凝胶是一种自足式预制胶（中型pH），通过将缓冲凝胶基质和电极装入一次性的、可透过紫外光的凝胶盒中制成。该凝胶专为在同一块水平凝胶中实现快速的、少缓冲液的、高通量的蛋白质电泳而设计，具有96孔、6%和48孔、8%两种规格。每块E-PAGE 96 6%凝胶含有96个样品泳道和8个蛋白标准品泳道，其独特的交错式孔板能兼容标准96孔板格式，可使用多通道移液器或自动化液体处理系统进行上样。每块E-PAGE 48 8%凝胶含有48个样品孔和4个蛋白标准品泳道。可使用多通道移液器在交替的泳道中上样，或使用自动化液体处理系统上样。

Robotic scripts for running E-Gel 96 Agarose Gels and E-PAGE 96 Gels on Beckman Coulter's Biomek FX workstation can be found on our website at: www.thermofisher.com/egels (click the Labware Definitions link in the left navigation pane).

Find additional tips, troubleshooting help, and resources within our Nucleic Acid Purification and Analysis Support Center.

The EG program is to run E-Gel 96 and 48 gels, while the EP is to run the E-PAGE 96 and 48 gels.

Find additional tips, troubleshooting help, and resources within our Nucleic Acid Purification and Analysis Support Center.

NuPAGE Transfer Buffer with 10% methanol provides optimal transfer of E-PAGE gels in the XCell II Blot Module. The NuPAGE Antioxidant is used in the transfer buffer for blotting reduced proteins and prevents the proteins from re-oxidizing. The Tris-Glycine Transfer Buffer has not been tested and it is not known as to what the transfer efficiencies would be like.

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

If you observe high background after Coomassie staining/destaining, here are some things to check:

It is very important to use 0.015% Coomassie R-250. Even using 0.03% will make Coomassie destaining more difficult.

The gel shouldn't be left in stain for more than 1 hour.

Increasing the destain time may reduce background.

Warming the gel and destain solution is also very important for best results.

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

The E-Editor 2.0 software allows you to quickly reconfigure digital images of E-Gel 48, E-Gel 96, E-PAGE 48 and E-PAGE 96 gel results for analysis and documentation. You can capture an image of the gel and then use the E-Editor 2.0 software to:
-Align and arrange the lanes in the image to any 48, 96, or 384 image
-Save the reconfigured image for further analysis
-Copy and paste selected lanes or the entire image into other applications for printing, saving, e-mailing, and/or publishing on the Web.

The E-Editor 2.0 does not take perform densitometry analysis from your gel images. The E-Editor 2.0 can be downloaded for free.

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

Biomek FX does not support the use of E-Gel 48 or E-PAGE 48, as the 8-span head is perpendicular to the alignment of the lanes. There is a 90 degree adaptor that may be made to make this work. Please contact Technical Support (contact information is on the Thermo Fisher Scientific website).

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To run E-PAGE gels, make sure the program EP is selected on the Mother E-Base. The recommended run time for E-PAGE 96, 6% gels is 14 min. The recommended run time for E-PAGE 48, 8% gels is 23 min. No pre-run is necessary.

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The Mother E-Base and Daughter E-Base can be used for either, E-PAGE 96, E-PAGE 48, E-Gel 96, or E-Gel 48 systems. However, the old E-Gel 96 motherbase and E-Gel 96 daughter base will not support the E-PAGE system and have been discontinued.

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The following protein MW standards are recommended for E-PAGE 96 gels.

Cat. No. LC5700 E-PAGE SeeBlue Pre-Stained Standard for general electrophoresis
Cat. No. LC5701 E-PAGE MagicMark Unstained Protein Standard for Western blotting
Cat. No. LC5928 BenchMark Fluorescent Protein Standard for fluorescent detection

The following protein MW standards are recommended for E-PAGE 48 gels.

Cat. No. LC5925 SeeBlue Plus 2 Pre-Stained Standard for general electrophoresis
Cat. No. LC5602 MagicMark XP Western Protein Standard for Western blotting
Cat. No. LC5928 BenchMark Fluorescent Protein Standard for fluorescent detection

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

Samples containing high salt or detergents will cause loss of resolution on E-PAGE 96 and E-PAGE 48 gels.

For E-PAGE 48, dilute the samples such that the final concentration of the salt or detergent in the samples is as described:
Triton X-100 <0.3%
Tween 20 <0.3%
Tris <300 mM
NaCl <300 mM
CHAPS <0.3%
NP-40 <0.3%
RIPA <0.25X
ammonium sulfate <100 mM
sodium acetate <200 mM
NuPAGE Sample Buffer <0.5X
SDS not recommended to add >2% as this is already in the loading buffer
EDTA <20 mM
MES is not recommended
Buffers containing glycine, DTT, imidazole, and urea are ok up to 500 mM.

For E-PAGE 96, dilute the samples such that the final concentration of the salt or detergent in the samples is as described: Triton X-100 <0.5%, Tween 20 <0.5%, SDS <4%, Tris <200 mM, NaCl <250 mM.

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

For SDS-PAGE and staining or blotting, we recommend using the 4X E-PAGE Loading Buffer 1 for preparing samples. We do not recommend using any other SDS-PAGE sample buffer. The E-PAGE Loading Buffer 1 is optimized for E-PAGE gels. If the sample is already in NuPAGE LDS sample buffer or the Tris-Glycine SDS sample buffer, it is fine to run on the E-PAGE gels. However, there may be a loss of resolution in the bands.

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Due to the thickness of the E-PAGE gels, they are not compatible with the iBlot3 device for transfer. Recommended methods for transfer of E-PAGE gels are semi-dry blotting or semi-wet blotting in the XCell II Blot Module. Methods are described in the E-PAGE Technical Guide.

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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.

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For semi-dry transfer of E-PAGE 48 gels, we recommend using 2X NuPAGE Transfer Buffer containing 15% methanol and for E-PAGE 96 gels, we recommend using 2X NuPAGE Transfer Buffer without methanol. Transfer at 25 V (constant) for 30-60 minutes. Please refer to Page 33 of the E-PAGE Technical Guide (http://tools.thermofisher.com/content/sfs/manuals/epagetechguide_man.pdf).

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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.

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.

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.

We recommend keeping the surfaces of the Mother E-Base Device and Daughter E-Base Device free of contaminants. To clean, disconnect bases from power source and wipe with a dry cloth. Do not attempt to open or service the bases.

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The E-Holder Platform is designed to hold E-PAGE Gels during loading. You can use the E-Holder Platform when you need to load multiple gels while other gels are running on the E-Base device.

Note: The E-Holder Platform is not a power supply unit, cannot be connected to an electrical outlet, and cannot be used to run E-PAGE 48 or 96 Gels. To obtain the best results, run E-PAGE Gels on the Mother E-Base Device or Daughter E-Base Device within 15 minutes after loading on the E-Holder Platform.

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

We recommend using the EP program for running E-PAGE gels.

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

E-PAGE gels are compatible with many standard Coomassie staining, silver staining, or fluorescent staining protocols. E-PAGE gels are thicker than most SDS-PAGE mini-gels, so additional time may be required for staining and destaining steps. We recommend the following stains for E-PAGE Gels. Detailed staining protocols are described on Pages 40-49 of the E-PAGE Technical guide (http://tools.thermofisher.com/content/sfs/manuals/epagetechguide_man.pdf).

Total protein stains:
*SYPRO Ruby Protein Gel Stain (Page 40)
*Coomassie Stain (Page 42)
*SimplyBlue SafeStain (Page 44)
*SilverQuest Silver Staining Kit (Page 47)
*SilverXpress Silver Staining Kit (Page 48)

Specific protein stains:
*Lumio Green Detection Reagent for detecting Lumio fusion proteins Ppage 40)
*InVision His-tag In-Gel Stain for detecting 6X His-tagged proteins (blotting recommended first, Page 49)

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

After electrophoresis is completed, the E-PAGE gel cassette can be opened with a gel knife to perform downstream applications such as western blotting or staining.

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Electrophoresis:
For E-PAGE 48 8% gels, use SeeBlue Plus 2 Pre-Stained Standard, Cat. No. LC5925.
For E-PAGE 96 6% gels, use E-PAGE SeeBlue Pre-Stained Standard, Cat. No. LC5700.

Western Blotting:
For E-PAGE 48 8% gels, use MagicMark XP Western Protein Standard, Cat. No. LC5602.
For E-PAGE 96 6% gels, use E-PAGE MagicMark Unstained Protein Standard, Cat. No. LC5701.

Fluorescence detection:
For E-PAGE 48 8% gels, use BenchMark Fluorescent Protein Standard, Cat. No. LC5928.
For E-PAGE 96 6% gels, use BenchMark Fluorescent Protein Standard, Cat. No. LC5928.

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

The recommended run time for E-PAGE 96 6% gels is 14 minutes and for E-PAGE 48 8% gels is 25 minutes. No pre-run is necessary. It is possible to run longer than the recommended run time to improve resolution, keeping in mind the risk of running into the next well below. Avoid running E-PAGE 96 or 48 gels for more than 25 and 30 minutes respectively. Running power is 9.5 watts.

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We recommend using the Large Gel Drying Kit (Cat. No. NI2207) for drying E-PAGE gels. It is also possible to air dry E-PAGE gels. The E-PAGE 96 gels will need at least 4 days for complete air drying. Vacuum drying is not recommended - the thickness of these gels makes them especially prone to cracking.

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Please follow the guidelines mentioned on Page 10 of the E-PAGE Technical Guide (http://tools.thermofisher.com/content/sfs/manuals/epagetechguide_man.pdf). Samples containing high salt or detergents result in loss of resolution on E-PAGE Gels.

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

The composition of the 4X E-PAGE Loading Buffer 1 is proprietary

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We recommend using the Lumio gel sample buffer that is supplied with the Lumio Green Detection kit, for in-gel detection. We do not recommend using the E-PAGE loading buffer. There is no need to remove the E-PAGE gels from the cassette to visualize Lumio fusion proteins.

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We recommend using the 4X E-PAGE Loading Buffer 1 (supplied with E-PAGE gels) for preparing samples for SDS-PAGE and staining or blotting using E-PAGE gels. We do not recommend using any other SDS sample buffer such as the NuPAGE LDS sample buffer or the Tris-Glycine SDS sample buffer as there may be a loss of resolution in the bands.

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The recommended total sample loading volume for E-PAGE 48 and E-PAGE 96 gels is 15 µL (up to 20 µL). Very low volumes of sample loaded will result in poor resolution and smearing. For proper band separation, we recommend keeping sample volumes uniform and loading deionized water into the empty wells.

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We recommend loading 1-20 ?g protein per lane of an E-PAGE gel. The maximum recommended protein load per lane gel is 20 µg. Excess protein will cause poor resolution and smearing.

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E-PAGE gels contain SDS and are recommended for performing electrophoresis under denaturing conditions.

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The running distance of each lane is 1.6 cm for E-PAGE 96 6% and 3.2 cm for E-PAGE 48 8% gels.

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The molecular weight range of proteins that can be separated is 10-220 kDa for E-PAGE 96 6% gels and 10-200 kDa for E-PAGE 48 8% gels.

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E-PAGE gels do not contain a preservative. They undergo UV sterilization during the production process.

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The gel formulation of E-PAGE gels is proprietary.

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E-PAGE gels are self-contained, pre-cast gels (neutral pH) that include a buffered gel matrix and electrodes packaged inside a disposable, UV-transparent cassette. They are designed for fast, buffer-less, high-throughput protein electrophoresis in a horizontal format and are available in 96-well, 6% and 48-well, 8% formats. Each E-PAGE 96 6% gel contains 96 sample lanes and 8 marker lanes in a patented staggered well-format that is compatible with the standard 96-well plate format for loading with a multichannel pipette or with an automated liquid handling system. Each E-PAGE 48 8% gel contains 48 sample wells and 4 marker wells. The wells are compatible for loading with a multichannel pipette in alternating lanes or with an automated liquid handling system.

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