ThermoFisher Scientific E-PAGE Gels Guía de inicio rápido

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.

For support visit www.lifetechnologies.com/support or email [email protected]om

www.lifetechnologies.com

06 October 2021

iii

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™Gels ............................................................................................................13

Running E-PAGE™Gels.............................................................................................................................17

Guidelines for Loading E-PAGE™Gels by Automated Liquid Handling ..........................................18

Using E-Holder™Platform ........................................................................................................................20

Opening the E-PAGE™Cassette ...............................................................................................................24

Blotting E-PAGE™Gels.........................................................................................................25

Blotting E-PAGE™Gels with the iBlot®Device ......................................................................................25

Semi-Dry Blotting of E-PAGE™Gels........................................................................................................31

Semi-Wet Blotting of E-PAGE™Gels .......................................................................................................35

Visualizing and Staining of E-PAGE™Gels ........................................................................38

Visualizing LumioFusion Proteins .......................................................................................................39

SYPRO®Ruby Staining Protocol ..............................................................................................................40

Coomassie Staining ....................................................................................................................................41

SimplyBlue™ SafeStain ...............................................................................................................................43

Silver Staining Methods.............................................................................................................................45

InVision™His-tag Protein Gel Stain.........................................................................................................48

Drying of E-PAGE™Gels ......................................................................................................49

Gel Drying ...................................................................................................................................................49

Analysis and Troubleshooting ............................................................................................51

Molecular Weight Calibration ..................................................................................................................51

Expected Results.........................................................................................................................................53

Using E-Editor™2.02 Software..................................................................................................................60

Troubleshooting..........................................................................................................................................61

Appendix....................................................................................................................................63

Additional Staining Protocols...................................................................................................................63

Product Specifications................................................................................................................................67

Accessory Products ....................................................................................................................................70

Technical Support.......................................................................................................................................72

Product Contents

Purpose of the

Guide

The E-PAGE™Technical Guide contains information about E-PAGE™Gels and

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™ Electrophoresis Device,

and are available in 48-well or 96-well formats. See pages 3–4 for further

specifications. See page 71 for ordering information.

Product Quantity Catalog no.

E-PAGE™48 8% Gels 8-pack EP048-08

E-PAGE™96 6% Gels 8-pack EP096-06

Kit Contents

Each pack of E-PAGE

™

Gels contain the following contents:

E-PAGE™Gel 8-Pack Contents Quantity

E-PAGEGels 8 gels

E-PAGELoading Buffer 1 (4X) 4.5 mL

E-PAGEBlotting Pad 1

Shipping and

Storage

The E-PAGE™48 and 96 Gels, E-PAGEBlotting Pad and Mother E-Base™are

shipped and stored at room temperature.

Do not allow the temperature to drop below 4°C or rise above 40°C when

storing the gels.

The E-PAGELoading Buffer is shipped at room temperature. Store the

E-PAGE™Loading Buffer 1 (4X) at room temperature or at 4°C.

Introduction

E-PAGE™Protein Electrophoresis System

About the

E-PAGE™Protein

Electrophoresis

System

The E-PAGE™Protein Electrophoresis System is designed for fast, medium-to-

high-throughput protein electrophoresis in a horizontal format.

The self-contained E-PAGE™48 or 96 Pre-cast Gels consist of a buffered gel

matrix and electrodes packaged inside a disposable, UV-transparent cassette.

The gels can be loaded by multichannel pipetter or automated loading system.

Electrophoresis of the samples is performed with the E-Base™Electrophoresis

Device. The E-Base™Electrophoresis Device consists of a base for

electrophoresis, and an integral power supply. After electrophoresis is

completed, the cassette can be opened with the Invitrogen™Gel Knife (Cat.

No. EI9010, sold separately) to perform downstream applications such as

western blotting or staining.

The E-Editor™2.02 software allows digital images of the E-PAGE™Gels to be

reconfigured into a side-by-side format for easy comparison and analysis.

Applications

The E-PAGE™Protein Electrophoresis System is ideal for screening protein

samples using the following applications:

•In-gel staining with Lumio™Green Reagent

•Western blotting

•Staining (Coomassie, silver, or fluorescent stains)

•Functional assays

Advantages

Using E-PAGE™Gels for protein analysis offers the following advantages:

•Produces fast, safe, and consistent electrophoresis

•Eliminates the need to prepare gels and buffers

•Compatible with most commercially available automated liquid handling

systems for high-throughput electrophoresis

•Available in two well formats to suit your applications

•Easy to open gel cassettes facilitate downstream staining and blotting

applications

•Ability to track samples during electrophoresis of multiple gels using an

individual barcode on each gel cassette

E-PAGE™Protein Electrophoresis System, Continued

Choosing a Gel

for your

Application

To obtain the best results for your application it is important to choose the

correct E-PAGE™Gel. The table below describes the major features of each

format. For specifications and separation range of each gel type, see page 67–68.

E-PAGE™ Gel No. Rows No. Wells Well Volume Run

Length

Run

Time

48 Gel 2* 48 sample

4 marker

15 µL

15 µL 3.2 cm 25 min.

96 Gel 8* 96 sample

8 marker

20 µL

20 µL 1.6 cm 14 min.

*Wells compatible for loading with a multichannel pipetter or with an automated

liquid handling system.

Western Blotting

of E-PAGE™Gels

E-PAGE

™

Gels are suitable for western blotting. The iBlot

Gel Transfer Device

is recommended method for blotting E-PAGE™Gels (page 25). Semi-dry

blotting (page 25), and semi-wet blotting may also be performed (page 35).

Tank blotting is not recommended, but can be performed if even pressure is

maintained between the gel and the membrane, and cooling is provided.

Staining E-PAGE™

Gels

E-PAGE

™

Gels are compatible with many standard Coomassie, silver, 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 38–48.

Total Protein Stains:

•SYPRO® Ruby Protein Gel Stain (page 38)

•Coomassie Stain (page 41)

•SimplyBlue™SafeStain (page 43)

•SilverQuest™ Silver Staining Kit (page 46)

•SilverXpress® Silver Staining Kit (page 47)

Specific Protein Stains:

•Lumio™Green Detection Reagent for detecting Lumiofusion proteins

(page 38)

•InVision™His-tag In-Gel Stain for detecting 6X His-tagged proteins

(blotting recommended first, page 48)

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)

•E-PAGE™ Blotting Pad

•E-Editor™2.02 Software

Details on each system component are described in this section.

E-PAGE48 Gels

•

E-PAGE™48 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™48 Gel contains 48 sample lanes and 4 marker lanes. This

configuration provides a 3.2-cm run length. The wells of E-PAGE™48 Gels

are compatible for loading with a multichannel pipetter in alternating lanes

or with an automated liquid handling system (see page 17 for automation

specifications).

•After electrophoresis, the E-PAGE™48 cassette is easily opened with the

Invitrogen™Gel Knife (Cat. No. EI9010, sold separately) to remove the gel

for staining or blotting applications.

•Each E-PAGE™48 cassette is labeled with an individual barcode to facilitate

identification of the gel using commercial barcode readers (page 13).

See page 67 for E-PAGE™48 8% Gel specifications.

Diagram of an

E-PAGE



Cassette

A diagram of the E-PAGE

™

48 Gel cassette is shown below.

Plastic combs

Copper

electrode (-)

Copper

electrode (+)

Barcode

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. The wells

of the E-PAGE™96 gels are compatible for loading with a multichannel

pipetter or with an automated liquid handling system (See page 17 for

automation specifications).

•After electrophoresis, the E-PAGE™cassette is easily opened with the

Invitrogen™Gel Knife (Cat. No. EI9010, sold separately) to remove the gel

from the cassette for staining or blotting applications.

•Each E-PAGE™96 cassette is labeled with an individual barcode to facilitate

identification of the gel using commercial barcode readers (page 13).

See page 67 for E-PAGE™96 6% Gel specifications

Diagram of an

E-PAGE™96

Cassette

A diagram of the E-PAGE™96 Gel cassette is shown below.

Copper

electrode (+)

Copper

electrode (-)

Barcode

Plastic combs

E-PAGE™System Components, Continued

E-Base

Electrophoresis

Device

E-PAGE™Gels are used with a specially designed electrophoresis device that

combines a base and a power supply. Two types of devices are available from

Life Technologies:

•The Mother E-Base™(Cat. no. EB-M03) has an electrical plug that can be

connected directly to an electrical outlet and is used for electrophoresis of

one E-PAGE™Gel. The Mother E-Base™has been tested for electrophoresis

with up to three Daughter E-Bases™connected at one time.

•The Daughter E-Base™(Cat. no. EB-D03) connects to the Mother E-Base™,

and together they can be used for the independent electrophoresis of 2 or

more E-PAGE™Gels. The Daughter E-Base™does not have an electrical plug

and cannot be used without a Mother E-Base™.

E-PAGE

™

Gels are not compatible with the E-Gel

96 bases

(Cat nos. G7100-01/G7200-01) previously available from Life Technologies. The

older E-Gel®96 bases do not have the ‘E-Base™’ inscription on the platform.

Mother E-Base™

Each Mother E-Base

™

has a pwr/prg (power/program) button (right side) and a

time button (left side) on the lower-right side of the base. The lower-left side of

each Mother E-Base™contains a light LED and a digital display. The gel cassette

is inserted into the two electrode connections. The Mother E-Base™is connected

to an electrical outlet with the electrical plug.

The E-Base™EP program is set to 14 minutes default time. See page 15 to adjust

time as recommended.

Mother E-Base



(–)

(+)

Pwr/prg button

Time button

Digital

display

To electrical outlet

LED Light

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

E-Base™(already connected to a Mother E-Base™). Once connected to a Mother

E-Base™, each Daughter E-Base™ is designed to function independently of the

Mother E-Base™ or other Daughter E-Bases™

Mother E-Base™/Daughter E-Base™

Daughter

-Base™

Mother

-Base™

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