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Related Manuals for ThermoFisher Scientific E-PAGE Gels
Summary of Contents for ThermoFisher Scientific E-PAGE Gels
- Page 1 E-PAGE Technical Guide ™ General information and protocols for using E-PAGE Gels ™ Catalog Number EP096-06 Revision B.0 Publication Number MAN0000374 Publication Part Number 25-0644 For Research Use Only. Not for use in diagnostic procedures.
- Page 2 For support visit www.lifetechnologies.com/support or email techsupport@lifetech.com www.lifetechnologies.com 06 October 2021...
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Page 3: Table Of Contents
Contents Product Contents ............................iv Introduction ..........................1 E-PAGE ™ Protein Electrophoresis System ....................1 E-PAGE ™ System Components ........................3 Methods ............................8 Electrophoresis of E-PAGE ™ Gels ..................8 Guidelines for Sample Preparation ......................8 Electrophoresis of E-PAGE ™... -
Page 4: Product Contents
Product Contents Purpose of the The E-PAGE ™ Technical Guide contains information about E-PAGE ™ Gels and Guide is intended to supplement the manuals supplied with E-PAGE ™ Gels. Details for sample preparation, electrophoresis conditions, blotting, and staining are included in this guide. Types of Kits E-PAGE gels are designed for use with the E-Base... -
Page 5: Introduction
Introduction E-PAGE Protein Electrophoresis System ™ About the The E-PAGE Protein Electrophoresis System is designed for fast, medium-to- ™ E-PAGE ™ Protein high-throughput protein electrophoresis in a horizontal format. Electrophoresis The self-contained E-PAGE 48 or 96 Pre-cast Gels consist of a buffered gel ™... - Page 6 E-PAGE Protein Electrophoresis System, ™ Continued Choosing a Gel To obtain the best results for your application it is important to choose the for your correct E-PAGE Gel. The table below describes the major features of each ™ Application format. For specifications and separation range of each gel type, see page 67–68. E-PAGE ™...
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Page 7: E-Page ™ System Components
E-PAGE System Components ™ Introduction The E-PAGE ™ Protein Electrophoresis System consists of the following components: • E-PAGE 48 or 96 Pre-cast Gels ™ • E-Base Electrophoresis Device ™ • E-Holder Platform ™ • E-PAGE Loading Buffer 1 (4X) ™ •... - Page 8 E-PAGE System Components, ™ Continued E-PAGE 96 Gels • E-PAGE 96 Gels are self-contained, pre-cast gels that include a buffered ™ gel matrix and electrodes packaged inside a disposable, UV-transparent cassette. • Each E-PAGE ™ 96 Gel contains 96 sample lanes and 8 marker lanes in a patented staggered well format that provides a 1.6-cm run length.
- Page 9 E-PAGE System Components, ™ Continued E-Base E-PAGE ™ Gels are used with a specially designed electrophoresis device that Electrophoresis combines a base and a power supply. Two types of devices are available from Life Technologies: Device • The Mother E-Base ™...
- Page 10 E-PAGE System Components, ™ Continued Daughter E-Base ™ The Daughter E-Base ™ is similar to the Mother E-Base ™ except the Daughter E-Base does not have an electrical cord and cannot be connected to an electrical ™ outlet. The Daughter E-Base is connected to a Mother E-Base or to another Daughter ™...
- Page 11 E-PAGE System Components, ™ Continued E-PAGE ™ Loading E-PAGE ™ Gels are supplied with E-PAGE ™ Loading Buffer 1 (4X), which is Buffer optimized for E-PAGE Gels, and is recommended for routine SDS-PAGE and ™ staining or blotting applications. It is not recommended to use any other SDS-PAGE sample buffer.
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Page 12: Methods
Methods Electrophoresis of E-PAGE Gels ™ Guidelines for Sample Preparation Introduction For optimal results using the E-PAGE ™ System, follow the guidelines for preparing your protein samples as described in this section. We recommend that you read this section before preparing your samples. Use the sample preparation method and loading buffer appropriate for your detection method: Application... - Page 13 Guidelines for Sample Preparation, Continued Total Sample For best results, avoid loading less than 5 µL of sample in wells, and maintain a Volume uniform loading volume. If you do not have enough samples to load all the wells of the gel, load an equal volume of deionized water into any empty wells. E-PAGE ™...
- Page 14 Guidelines for Sample Preparation, Continued Loading Buffer Based on your application, use the appropriate loading buffer as described below: • SDS-PAGE and routine staining (Method 1, page 11) For SDS-PAGE and routine staining or blotting, use the E-PAGE ™ Loading Buffer 1 (4X) included in the kit for preparing samples.
- Page 15 Guidelines for Sample Preparation, Continued Method 1: Routine Use this protocol if you are performing SDS-PAGE followed by routine staining Staining and or blotting. Blotting If the E-PAGE Loading Buffer 1 (4X) is stored at 4°C, bring the buffer to room ™...
- Page 16 Guidelines for Sample Preparation, Continued Method 2: Lumio ™ A brief protocol to prepare samples for specific detection of Lumio ™ fusion Detection proteins using the Lumio Green Detection Kit is described below. ™ For details on the Lumio ™ Green Detection Kit, refer to the manual available at www.lifetechnologies.com or contact Technical Support (page 72).
- Page 17 Electrophoresis of E-PAGE Gels ™ Introduction After preparing your samples, you are ready to load E-PAGE ™ Gels. This section describes the procedure for loading protein samples and molecular weight standards. The Mother E-Base and Daughter E-Base Electrophoresis Devices are ™...
- Page 18 Electrophoresis of E-PAGE Gels, ™ Continued • Always load 5–10 µL deionized water first into all wells prior to loading sample or molecular weight standard. • E-PAGE ™ Gels can only be used once. Do not re-use. • To obtain the best results, run the E-PAGE ™...
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Page 19: Electrophoresis Of E-Page
Electrophoresis of E-PAGE Gels, ™ Continued Selecting a The recommended program for E-PAGE ™ Gels is EP. Select program EP prior Program on the to inserting a gel into the base. E-Base ™ Device Brief instructions for using the E-Base Device are included in this manual. - Page 20 Electrophoresis of E-PAGE Gels, ™ Continued Loading Gels Each E-PAGE ™ Gel is individually wrapped and ready for use. Refer to pages 17–19 for automated liquid handling system guidelines for loading E-PAGE ™ Gels. To load your samples on the gels using the E-Holder ™...
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Page 21: Running E-Page ™ Gels
Running E-PAGE Gels ™ Introduction After loading your protein samples on the E-PAGE ™ Gels, proceed immediately to electrophoresis using the E-Base ™ Device. Refer to page 22 for the E-Base Quick Reference Guide. ™ Using E-Base ™ Instructions for running E-PAGE ™... -
Page 22: Guidelines For Loading E-Page ™ Gels By Automated Liquid Handling
Guidelines for Loading E-PAGE Gels by Automated Liquid ™ Handling Introduction E-PAGE 48 and 96 Gels are designed for automated loading using standard SBS (Society for Biomolecular Screening)-compatible liquid handling systems with an 8, 12, or 96-tip robotic loading apparatus. In general, follow the manufacturer instructions for your automated loading system. - Page 23 Guidelines for Loading E-PAGE Gels by Automated Liquid ™ Handling, Continued Automated E-PAGE 48 Gels are compatible with any automated liquid handling system Loading of that has an 8-span loading head with either fixed (9-mm) or variable distance E-PAGE ™...
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Page 24: Using E-Holder ™ Platform
Using E-Holder Platform ™ Introduction 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 ™... - Page 25 Running E-PAGE Gels, ™ Continued Using E-Base ™ 3. The Mother E-Base ™ Device or Daughter E-Base ™ Device signals the end of Continued the run with a flashing red light and rapid beeping for 2 minutes followed by a single beep every minute. At the end of the run, the digital display shows the original time setting.
- Page 26 E-Base Quick Reference Guide ™ Introduction A quick reference guide for operating the Mother E-Base ™ Device and Daughter E-Base Device is provided below. Operating modes and electrophoresis runs ™ are described. Mode Action Sound Light Digital Display Without gel cassette: Mother No light if a cassette last program used...
- Page 27 E-Base Quick Reference Guide, ™ Continued Mode Action Sound Light Digital Display Return to Ready Press and release mode after an the pwr/prg — Steady red Last time setting automatic stop button Press and release Restart after a manual the pwr/prg —...
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Page 28: Opening The E-Page ™ Cassette
Opening the E-PAGE Cassette ™ Opening To remove the E-PAGE ™ 48 or E-PAGE ™ 96 Gel from the cassette for blotting or E-PAGE ™ 48 or staining, use the Gel Knife to open the cassette. E-PAGE ™ 1. Separate each of the bonded sides of the E-PAGE ™... -
Page 29: Blotting E-Page ™ Gels
Blotting E-PAGE Gels ™ Blotting E-PAGE Gels with the iBlot Device ™ ® Introduction Instructions are provided in this section to assemble the iBlot Gel Transfer ® Device with the De-Bubbling Roller for blotting E-PAGE Gels. The ™ recommended program for blotting E-PAGE ™... - Page 30 Using the iBlot Device with the De-Bubbling Roller, ® Continued Assembling the 1. Open the lid of the device and pull up the Metal Spacers 1 and 2. If you iBlot ® Device have attached the De-bubbling Roller to the device, then remove the roller as shown in the figure below.
- Page 31 Using the iBlot Device with the De-Bubbling Roller, ® Continued Assembling the 4. Clean the Metal Spacer 1 with a damp cloth or tissue and place the spacer iBlot ® Device, on the membrane as shown below. continued Spacer 1 5.
- Page 32 Using the iBlot Device with the De-Bubbling Roller, ® Continued Assembling the 8. Insert the steel iBlot ® E-PAGE ™ Tab in the plastic tray groove with the tab iBlot ® Device, teeth facing up (figure A). Gently press the iBlot Cathode Stack over the ®...
- Page 33 Using the iBlot Device with the De-Bubbling Roller, ® Continued Assembling the 11. Hold the iBlot ® E-PAGE ™ Tab and plastic tab on the iBlot ® Anode Stack, iBlot ® Device, Bottom together and pull the assembly (anode and cathode stacks, and gel) continued together through the De-bubbling Roller towards the blotting surface, in one smooth, uninterrupted movement until the assembly reaches the Gel...
- Page 34 Using the iBlot Device with the De-Bubbling Roller, ® Continued Disassembling the To obtain good transfer and detection results, disassemble the device and stacks iBlot ® Gel Transfer within 30 minutes of ending the blotting procedure. Device 1. Open the lid of the iBlot ®...
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Page 35: Semi-Dry Blotting Of E-Page ™ Gels
Semi-Dry Blotting of E-PAGE Gels ™ Introduction A semi-dry blotting procedure for blotting E-PAGE ™ Gels is described in this section. You will need a semi-dry transfer apparatus that is capable of accommodating the dimensions of an E-PAGE Gel (8.6 cm x 13.5 cm) and a ™... - Page 36 Semi-Dry Blotting of E-PAGE Gels, Continued E-PAGE We recommend using 2X NuPAGE ® Transfer Buffer with 15% methanol and Transfer Buffer NuPAGE Antioxidant for the transfer of most proteins from E-PAGE ® ™ Gels. When the complete transfer of higher molecular weight proteins (>150 kDa) is desired, reduce methanol to 10%.
- Page 37 Semi-Dry Blotting of E-PAGE Gels, ™ Continued Equilibrating the Equilibration of the gel in 2X Transfer Buffer results in removal of salts that may increase conductivity and heat during transfer. Perform equilibration for the recommended time, as longer equilibration results in protein diffusion. 1.
- Page 38 Semi-Dry Blotting of E-PAGE Gels, ™ Continued Semi-Dry Blotting 1. In a clean container or Incubation Tray, soak 4 pieces of 2.5 mm Blotting Protocol Filter Paper (8.6 cm x 13.5 cm) in 2X NuPAGE Transfer Buffer (see ® page 32). Remove any air bubbles trapped between filter paper sheets using the Blotting Roller while the paper is still submerged in buffer.
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Page 39: Semi-Wet Blotting Of E-Page
Semi-Wet Blotting of E-PAGE Gels ™ Introduction A semi-wet blotting procedure using an XCell II ™ Blot Module for transfer of proteins from E-PAGE Gels is described below. ™ Semi-Wet Two XCell II ™ Blot Module units are required to transfer one E-PAGE ™... - Page 40 Semi-Wet Blotting of E-PAGE Gels, ™ Continued Preparing 1X We recommend using 1X NuPAGE ® Transfer Buffer with NuPAGE ® Transfer Buffer Antioxidant and 10% methanol for semi-wet transfer of proteins from the E-PAGE ™ Gel. Note: If a large amount of heat is produced during the transfer, use a cooling unit or perform transfer with cold buffer.
- Page 41 Semi-Wet Blotting of E-PAGE Gels, ™ Continued Semi-Wet 1. Remove any small pieces of gel from the well areas by gently rubbing a Blotting gloved finger over the well side of the gel. The gel pieces on the gel may Procedure cause air bubbles and field distortion during transfer.
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Page 42: Visualizing And Staining Of E-Page
Visualizing and Staining of E-PAGE Gels ™ Choosing a A summary of the time needed and sensitivity obtained with E-PAGE gels ™ Staining Protocol using various staining protocols is described below. Based on the starting material and your requirements for sensitivity and background, choose an appropriate staining protocol. -
Page 43: Visualizing Lumio Fusion Proteins
Visualizing Lumio Fusion Proteins Introduction The steps involved in detecting Lumio ™ fusion proteins run on an E-PAGE ™ are described below. To visualize Lumio ™ fusion protein bands after electrophoresis, you will need a UV transilluminator or a laser-based scanner (see below). For further details on imaging Lumio ™... -
Page 44: Sypro ® Ruby Staining Protocol
SYPRO Ruby Staining Protocol ® Introduction Instructions for staining E-PAGE ™ Gels using SYPRO ® Ruby Protein Gel Stain is described in this section. To visualize protein bands after electrophoresis using SYPRO Ruby, you will need a UV transilluminator or a laser-based scanner (see ®... -
Page 45: Coomassie Staining
Coomassie Staining Introduction Instructions for staining E-PAGE ™ Gels using Coomassie R-250 are described in this section. To obtain maximum sensitivity, the total staining time for Coomassie R-250 staining is 1.5 h plus overnight destaining. The use of the Coomassie R-250 microwave protocol reduces the amount of time needed for staining and destaining, however for minimal background, overnight destaining is recommended. - Page 46 Coomassie Staining, Continued The staining solutions for the Coomassie R-250 staining protocol and the microwave staining protocol are different. Be sure to use the correct stain for the correct protocol. Coomassie ® For all staining and destaining steps described below, be sure to use sufficient R-250 Staining reagents to completely cover the gel using a suitable container such that the gel Protocol...
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Page 47: Simplyblue ™ Safestain
SimplyBlue SafeStain ™ Introduction Instructions for staining E-PAGE ™ Gels using SimplyBlue ™ SafeStain are described in this section. For further details, refer to the product manual available at www.lifetechnologies.com or contact Technical Support (page 72). The total staining time for SimplyBlue ™... - Page 48 SimplyBlue SafeStain, ™ Continued E-PAGE 96 Gels For routine staining of E-PAGE 96 Gels, use Protocol A. To obtain the clearest background for imaging, use Protocol B, which includes a 12–24 hour washing step to improve the background. Protocol A 1.
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Page 49: Silver Staining Methods
Silver Staining Methods Introduction Instructions for staining E-PAGE ™ Gels using SilverQuest ™ Silver Staining Kit or SilverXpress Silver Staining Kit are described in this section. ® Total staining time with SilverQuest ™ Silver Staining Kit is 4.5 hours and SilverXpress ®... - Page 50 Silver Staining Methods, Continued SilverQuest ™ A brief protocol for staining one E-PAGE ™ Gel with the SilverQuest ™ Silver Silver Staining Staining Kit is described below. For details, refer to the product manual Protocol available at www.lifetechnologies.com or contact Technical Support (page 72). After electrophoresis, remove the gel from the cassette (page 24).
- Page 51 Silver Staining Methods, Continued SilverXpress ® A brief protocol for staining one E-PAGE ™ Gel with the SilverXpress ® Silver Silver Staining Staining Kit is described below. For details, refer to the product manual available Protocol at www.lifetechnologies.com or contact Technical Support (page 72). After electrophoresis, remove the gel from the cassette (page 24).
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Page 52: Invision ™ His-Tag Protein Gel Stain
InVision His-tag Protein Gel Stain ™ Introduction For detection of His-tagged proteins, use the following protocol. Due to the thickness of E-PAGE Gels, we recommend transferring proteins onto nitrocellulose membrane (see pages 25-37) and then detecting them using InVision His-tag In-Gel Stain. -
Page 53: Drying Of E-Page ™ Gels
Drying of E-PAGE Gels ™ Gel Drying Introduction We recommend drying E-PAGE Gels using passive air-drying methods such ™ as the Large Gel Drying Kit. Due to the thickness of E-PAGE ™ Gels, vacuum drying is not recommended and may cause the gel to crack. Materials Needed ... - Page 54 Gel Drying, Continued Gel Drying 7. Place the solid square of the Large Gel Drying Frame on a paper towel with the Protocol, corner pins facing up. continued 8. Lay a piece of pre-wetted cellophane from Step 5 over the frame carefully without trapping any air bubbles.
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Page 55: Analysis And Troubleshooting
Analysis and Troubleshooting Molecular Weight Calibration Molecular Weight The molecular weight of a protein can be determined based upon its relative Calibration mobility by constructing a standard curve with protein standards of known molecular weights. The protein mobility in SDS gels is dependent on: •... - Page 56 Molecular Weight Calibration, Continued Assigned Apparent MagicMark XP Western Protein Standard ™ Molecular Weights, Molecular Weight on E-PAGE ™ 48 8% Gel Continued 1. 209 kDa 2. 130 kDa 3. 114 kDa 4. 84 kDa 5. 64 kDa 6. 51 kDa 7.
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Page 57: Expected Results
Expected Results Western Blotting Results obtained using an E-PAGE ™ 48 8% Gel that was blotted using the semi-dry protocol described in this manual are shown below. MagicMark ™ XP Western Protein Standard (5 µL) was loaded in all sample and marker wells. - Page 58 Expected Results, Continued Lumio ™ Green Results obtained with an E-PAGE ™ 48 8% Gel using the Lumio ™ Green Detection Detection Kit are shown below. Various concentrations of a Lumio ™ fusion protein were labeled with Lumio ™ Green Detection Kit and run on an E-PAGE 48 8% Gel as described in this ™...
- Page 59 Expected Results, Continued SYPRO ® Ruby Results obtained using an E-PAGE ™ 48 8% Gel stained with SYPRO ® Ruby are Protein Gel Stain shown below. BSA (2.5 - 1000 ng) was run on an E-PAGE ™ 48 8% Gel and stained with SYPRO Ruby Protein Gel Stain as described in this manual.
- Page 60 Expected Results, Continued Coomassie ® R-250 Results obtained using an E-PAGE ™ 48 8% Gel stained with Coomassie R-250 Stain Stain are shown below. Samples were run on an E-PAGE ™ 48 8% Gel and stained with Coomassie R-250 Stain using the microwave protocol as described in this manual. M 1 2 3 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 M M 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 M...
- Page 61 Expected Results, Continued SimplyBlue ™ Results obtained using an E-PAGE ™ 48 8% Gel stained with SimplyBlue ™ SafeStain SafeStain are shown below. Samples were run on an E-PAGE ™ 48 8% Gel and stained with SimplyBlue SafeStain as described in this manual. ™...
- Page 62 Expected Results, Continued SilverQuest ™ Results obtained using an E-PAGE ™ 48 8% Gel stained with the SilverQuest ™ Silver Staining Kit Silver Staining Kit with are shown below. BSA (0.5–1000 ng) was run on an E-PAGE ™ 48 8% Gel and stained with the SilverQuest Silver Staining Kit as described in this manual.
- Page 63 Expected Results, Continued SilverXpress ™ Results obtained using an E-PAGE ™ 48 8% Gel stained with the Silver Staining Kit SilverXpress Silver Staining Kit with are shown below. ™ Samples were run on an E-PAGE ™ 48 8% Gel and stained with the SilverXpress ™...
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Page 64: Using E-Editor ™ 2.02 Software
Using E-Editor 2.02 Software ™ Introduction The E-Editor ™ 2.02 software for Windows ® allows you to reconfigure digital images of E-PAGE Gels for analysis and documentation. E-Editor ™ 2.02 software reconfigures the wells of the E-PAGE Gels into a side-by-side format for easy comparison and analysis. -
Page 65: Troubleshooting
Troubleshooting Troubleshooting The table below provides some solutions to possible problems you might encounter during the electrophoresis of E-PAGE Gels. For issues with protein ™ transfer, refer to the manual for the iBlot Gel Transfer Device. ® Observation Cause Solution No current Daughter E-Base Device used... - Page 66 Troubleshooting, Continued Observation Cause Solution Poor resolution or High salt or detergent concentration Be sure the final concentration of salt or smearing of bands in samples detergent in the sample is as described on page 9. You may need to manually increase the run time for high salt or detergent samples to obtain optimal results.
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Page 67: Appendix
Appendix Additional Staining Protocols Pro-Q Diamond ® Instructions for specific staining of phosphoproteins on E-PAGE Gels using ™ Phosphoprotein Pro-Q ® Diamond Phosphoprotein Gel Stain is described in this section. Gel Stain The total staining time is ~5 hours. Due to the thickness of E-PAGE ™... - Page 68 Additional Staining Protocols, Continued Phosphoprotein 5. Destain the gel with Pro-Q ® Diamond Phosphoprotein Gel destaining Staining Protocol, solution twice for 30 minutes each time at room temperature with gentle Continued shaking. You may wash the gel overnight to improve sensitivity and obtain clear background.
- Page 69 Additional Staining Protocols, Continued Pro-Q ® Sapphire Instructions for specific staining His-tagged proteins on E-PAGE ™ Gels using 532 Oligohistidine Pro-Q Sapphire 532 Oligohistidine Gel Stain is described in this section. ® Gel Stain The total staining time is ~5 hours. Due to the thickness of E-PAGE ™...
- Page 70 Additional Staining Protocols, Continued Oligohistidine 7. Place the gel on a UV transilluminator equipped with a standard camera and Staining select an appropriate filter on the camera (stain emission maxima is 572 nm). Procedure, You may also use a laser-based scanner with a laser line that falls within the Continued excitation maxima of the stain (532 nm).
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Page 71: Product Specifications
Product Specifications E-PAGE ™ 48 Gel Each E-PAGE ™ 48 gel contains 48 sample wells and 4 marker wells (M). Specifications 13.5 cm (l) × 10.8 cm (w) × 0.67 cm (thick) Cassette Size: Gel Thickness: 3.7 mm Gel Volume: 50 mL Gel Formulation: Proprietary, operating at a neutral pH... - Page 72 Product Specifications, Continued E-PAGE ™ Separation Range The migration and resolution range of proteins run on E-PAGE 48 8% Gels and E-PAGE 96 6% Gels are shown. E-Base ™ The specifications for the Mother E-Base and Daughter E-Base are listed ™...
- Page 73 Explanation of Symbols and Warnings E-Base ™ The Mother E-Base ™ and Daughter E-Base ™ comply with the Underwriters Laboratories Inc. regulation and the European Community Safety requirements. Operation of the E-PAGE bases is subject to the following conditions: •...
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Page 74: Accessory Products
Accessory Products E-PAGE ™ Gels The following E-PAGE ™ Gel kits are available from Life Technologies. Product Quantity Catalog no. E-PAGE 48 8% Gels 8-pack EP048-08 ™ E-PAGE 96 6% Gels 8-pack EP096-06 ™ Electrophoresis The following electrophoresis bases are available from Life Technologies for Bases running E-PAGE ™... - Page 75 Accessory Products, Continued Additional The following products for use with E-PAGE ™ gels are available separately from Products Life Technologies: Product Quantity Catalog no. SeeBlue ® Plus2 Pre-Stained Standard 500 µL LC5925 E-PAGE SeeBlue Pre-stained Protein Standard 500 µL LC5700 ™...
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Page 76: Technical Support
Technical Support Obtaining support For the latest services and support information for all locations, go to www.lifetechnologies.com At the website, you can: • Access worldwide telephone and fax numbers to contact Technical Support and Sales facilities • Search through frequently asked questions (FAQs) •...
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