ExcelGel 2-D Homogeneous 12 - VWR

ExcelGel 2-D Homogeneous 12.5

Polyacrylamide gel and buffer stripsfor flatbed SDS electrophoresis

Instructions

71-5009-03

Edition AA

Important Information

ExcelGel, Multiphor, MultiTempHoefer, and PlusOne are trade-marks of Amersham BiosciencesLimited or its subsidiaries.Amersham and Amersham Biosciencesis a trademark of Amersham plc.

©Amersham Biosciences AB1999 – All rights reserved

All goods and services are soldsubject to the terms and conditionsof sale of the company within theAmersham Biosciences groupwhich supplies them. A copy of theseterms and conditions is available onrequest.

Amersham Biosciences ABSE-751 84 UppsalaSweden

Amersham Biosciences UKLimitedAmersham Place, Little ChalfontBucks HP7 9NAEngland

Amersham Biosciences Inc800 Centennial Avenue, P.O.Box 1327Piscataway, NJ 08855USA

Amersham BiosciencesEurope GmbHPostfach 5480D-79021 FreiburgGermany

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Contents1. Introduction .............................................................. 2

1.1 Package contents and technical data .................. 21.2 Recommended equipment and accessories .......... 3

2. Use of ExcelGel 2-D Homogeneous 12.5in 2-D electrophoresis ............................................... 4

2.1 Equilibration of Immobiline DryStrip gels ......... 42.3 Applying the strip positioner .............................. 62.4 Applying buffer strips ........................................ 72.5 Applying Immobiline DryStrip gels .................... 82.6 Positioning the electrodes ................................. 102.7 Running conditions .......................................... 10

3. Detection ................................................................ 11

Contents

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1. Introduction

ExcelGel™ 2-D Homogeneous 12.5 is a 0.5-mm-thickprecast polyacrylamide gel for flatbed electrophoresis ofSDS denatured proteins, particularly for the seconddimension in 2-D electrophoresis separations. To facilitatehandling, the gel is cast on a plastic support film. The gelsize is 250 × 110 × 0.5 mm. A 6% acrylamide stackingzone on the cathodic side of the gel merges continuouslyinto a homogeneous 12.5% acrylamide separation zone.The gel is designed for use with ExcelGel SDS BufferStrips. These polyacrylamide strips contain all the bufferneeded for SDS electrophoresis and are supplied ready touse. The buffer in the strips together with the buffer in thegel constitute a discontinuous buffer system for improvedresolution.

The gel is recommended for use on the Multiphor™ IIElectrophoresis Unit.

These instructions describe the use of ExcelGel 2-DHomogeneous 12.5 for the second dimension separationin 2-D electrophoresis.

Please refer to Application Note 80-6443-47 “MultipleMini-format 2-D Electrophoresis on Multiphor II”, UserManual 80-6443-66 for Multiphor II Strip Positioner andhandbook 80-6429-60 “2-D Electrophoresis Using Immo-bilized pH Gradients - Principles and Methods” foradditional information.

Package contents

Each gel package contains 6 gels and instructions.

Code No. Designation No. per pack

17-6002-21 ExcelGel 2-D 6Homogeneous 12.5

71-5009-03 Instructions 1

1.1 Packagecontentsand techni-cal data

1. Introduction

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Technical data

Stacking gel zone 33 mm long, T=6%, C=3%

Separating gel zone 77 mm long, T=12.5%, C=2%

Separation range Mr 10,000-120,000

Gel dimensions: 250 × 110 × 0.5 mm

Gel buffer: Tris-acetate, pH 6.4

Gel backing: Polyester film

Shelf life: 15 months

Storage: +4 to +8 °C

Code No. Designation

17-1342-01 ExcelGel SDS Buffer Strips18-1018-06 Multiphor II Electrophoresis Unit80-6442-90 Multiphor II Buffer Strip Positioner80-1129-46 IEF Sample Application Pieces18-1123-96 EPS 1000 Power Supply, 1.0 kV19-3500-01 EPS 3500 XL Power Supply, 3.5 kV18-1102-77 MultiTemp™ III Refrigerated Bath

Circulator, 115 V18-1102-78 MultiTemp III Refrigerated Bath Circulator,

230 V80-6395-02 Hoefer™ Automated Gel Stainer with

19 × 29 cm PTFE-coated stainless steeltray.

Accepts gels up to 160 × 260 mm.80-6396-16 Hoefer Automated Gel Stainer with

29 × 35 cmPTFE-coated stainless steel tray.Accepts gels up to 280 × 260 mm.

17-1313-01 PlusOne™ SDS, 100 g17-1318-01 PlusOne DTT, 1 g17-1319-01 PlusOne Urea, 500 g17-1321-01 PlusOne Tris, 500 g17-1325-01 PlusOne Glycerol 87%, 1000 ml17-1329-01 PlusOne Bromophenol Blue, 10 g17-1150-01 PlusOne Silver Staining Kit, Protein17-0446-01 LMW Marker Kit

1.2 Recom-mendedchemicalsandaccessories

1. Introduction

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2. Use of ExcelGel 2-DHomogeneous 12.5 in2-D electrophoresisImportant: To avoid contaminating gels with spuriousprotein, always wear gloves when handling first dimen-sion strips and ExcelGel SDS gels.

Run and equilibrate the first dimension Immobiline™DryStrip gels [immobilized pH gradient (IPG) strips] asdescribed in 2-D Electrophoresis Using Immobilized pHGradients – Principles and Methods. Equilibrate the IPGstrips for 15 minutes in second dimension equilibrationsolution containing 1% (w/v) DTT followed by 15minutes more in equilibration solution containing 2.5%iodoacetamide. Three 7-cm long IPG strips can be equili-brated simultaneously in a 15 ml conical centrifuge tubeusing 10 ml of equilibration solution.

While the IPG strips are equilibrating for the seconddimension, prepare the Multiphor II unit for SDS electro-phoresis. Connect the Multiphor II electrophoresis unit tothe MultiTemp III thermostatic circulator and set thetemperature to 15 °C.

Pipette 1.5 ml of kerosene onto the Multiphor II coolingplate. Cut away the edges of the foil packaging for theExcelGel 2-D Homogeneous 12.5 gel and remove the gel.

A notch identifies the lower-left corner of the gel backingand the anodic (+) end of the gel. Markings on the plasticcover of the gel indicate the direction of electrophoresis.Orient the gel according to these markings and removethe cover of the Multiphor II unit.

2. Use of ExcelGel 2-D Homogeneous 12.5 in 2-D electrophoresis

2.1 Equili-bration ofImmobi-lineDryStripgels

2.2 PreparingtheMultiphor IIand placingtheExcelGel

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Carefully lower the gel onto the cooling plate so that thekerosene spreads completely under the gel backing. Thecathodic (–) edge of the gel should line up with the edgeof the grid on the cooling plate and the gel should becentred between the right and left edges of the coolingplate (Figure 1).

Note: Avoid trapping bubbles between the gel andthe cooling plate. Do not allow kerosene to touchthe gel surface.

To improve separation quality, allow the gel to dryslightly by leaving it uncovered for 5–15 minutes beforeturning on the MultiTemp III Thermostatic Circulator.


Fig. 1. ExcelGel on Multiphor II.

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2.3 Applyingthe strippositioner


Place the Multiphor II Buffer Strip Positioner over the geland cooling plate. The pegs protruding from the bottomof the Strip Positioner should be in contact with theshorter sides of the cooling plate. Use the (–) and (+)symbols to correctly orient the Strip Positioner (Figure 2).

Slide the Strip Positioner so that the cathodic (–) edge ofthe gel bisects the slot at position 1 (Figure 3). Lock theStrip Positioner in place by turning the grey locking camuntil the Strip Positioner cannot move.

Fig. 2. Placing the Strip Positioner.

Fig. 3. (drawing of slots 1 and 2, and the edge of the gel).

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2.4 Applyingthe bufferstrips


Note: Moisten your gloves with distilled waterbefore removing the buffer strips from their pack-aging. Always handle the buffer strips by their ends.

Carefully peel back the foil on the colourless cathodic (–)SDS buffer strip. Place it in the slot at position 1. Makesure that the buffer strip is aligned with the edge of theslot and that the narrow face of the buffer strip is down-ward (Figure 4).

Place a few drops of distilled water along the top face ofthe buffer strip. Stroke the buffer strip with a spatula orflat forceps to ensure complete contact with the cathodic(–) edge of the gel. The buffer strip should sit snuglywithin the slot (Figure 5).

Note: If a buffer strip breaks, put the pieces to-gether in the Strip Positioner on the gel surface.

Handle the yellow-coloured (+) anodic buffer strip in thesame way and place it in the slot at position 3 (the centreslot) of the Strip Positioner (Figures 4 and 5).

Fig. 4. Applying a buffer strip.

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Use forceps to remove the IPG strips from the equilibra-tion solution. Remove excess equilibration solution fromthe surface of the IPG strips by lightly tapping edgewiseonto a sheet of moistened filter paper. Alternatively, theIPG strips can be lightly and quickly blotted, gel sidedown, onto moistened filter paper. The IPG strips can beleft resting, gel side up, on the moistened filter paper forup to 10 minutes before proceeding to the next step.

Place the IPG strip(s) gel side down on the ExcelGelthrough the slot at position 2. The IPG strip(s) should liein the centre of the slot, parallel to the buffer strip, withapproximately 3 mm between the buffer strip and the IPGstrip(s). If multiple short IPG strips are being applied tothe same second dimension gel, the strips should be in asingle, straight line. Leave about at least 1 cm betweenthe ends of each IPG strip (Figure 6).

Use forceps to place one IEF sample application piece atthe ends of each IPG strip underneath the plastic tabformed by the overhanging gel support film at each end ofthe IPG strip. Be sure that the application pieces touch theends of the IPG strip (Figure 7).

2.5 Applyingthe equili-bratedIPG strips

Fig. 5. Seating the buffer strip in the slot on the gel.

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Note: Application pieces absorb any water thatflows out of the IPG strips during electrophoresis.

Make sure that the IPG strip is in full, direct contact withthe SDS gel. To remove any air bubbles, stroke the plasticbacking of the IPG strip gently with a spatula or a pair offorceps.

Fig. 6. Applying the IPG strip.

Fig. 7. Inserting application pieces at ends of IPG strips.

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To load marker proteins, place an extra application piece(or half piece) on the surface of the gel between two of theIPG strips, or just beyond the end of one of the outer IPGstrips. Pipette the marker proteins onto the extra sampleapplication piece. Apply the maker proteins in a volumeof 15 to 20 µl. For less volume, decrease the size of thesample application piece proportionally.

Place the IEF electrode holder on the electrophoresis unit,in the upper (non-contact) position, and align the elec-trodes with the centre of the buffer strips. Plug in theelectrode connectors and carefully lower the electrodeholder onto the buffer strips. See the Multiphor II UserManual page 22 and 23.

Place the Safety Lid on the Multiphor II unit. Connect thepower supply. Recommended electrical settings andrunning times are listed in Table 1.

Note: It is important to use a protocol with a low-current sample entry phase. Prior to the second,higher current phase (as indicated in footnote 1 ofTable 1), remove the IPG strips and applicationpieces and move the (–) cathodic buffer strip intothe slot in the positioner formerly occupied by theIPG strips (position 2). Ensure complete contact ofthe buffer strip with the gel again by placing a fewdrops of distilled water along the top surface of thebuffer strip and stroking with a spatula or flatforceps.

2.6 Position-ing theelectrodes

2.7 Runningconditions


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3. Detection

Table 1 . Recommended running conditions for one ExcelGel2-D Homogeneous 12.5.

Voltage Current Power DurationPhase (V) (mA) (W) (h:min)

1 600 20 30 ~0:351

2 600 50 30 ~1:152

1 When the bromophenol blue dye has moved beyond the slotcontaining the IPG strips, turn the power off and remove the IPGstrips and the application pieces from the gel. Then move thecathodic buffer strip into the slot in the Strip Positioner formerlyoccupied by the IPG strip(s) (position 2), covering the area of theremoved IPG strip(s). Ensure complete contact of the buffer stripwith the gel again by placing a few drops of distilled water along thetop surface of the buffer strip and stroking with a spatula or flatforceps. Adjust the position of the cathodic electrode, replace thesafety lid and turn the power back on.

2 Stop electrophoresis just as the bromophenol blue front reachesthe anodic buffer strip. Remove and discard the buffer strips.

Silver staining is recommended for ExcelGel 2-D Homo-geneous 12.5. The Hoefer Automated Gel Stainer auto-mates the gel staining process and, combined with the useof PlusOne Silver Staining Kit, Protein, eliminates most ofvariability associated with silver staining.


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ExcelGel 2-D Homogeneous 12 - VWR