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

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).

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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.

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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.

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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.

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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.

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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

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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.

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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.

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

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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 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.

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

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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)

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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.

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