Bio-Rad Whole Gel Eluter and Mini Whole Gel Eluter User Manual
Whole Gel Eluter
and
Mini Whole Gel Eluter
Instruction Manual
Catalog Numbers
165-1250
165-1251
165-1255
165-1256
For Technical Service Call Your Local Bio-Rad Office or in the U.S. Call 1-800-4BIORAD (1-800-424-6723)
Table of Contents
Page
Section 1 General Information....................................................................................1
1.1 Introduction ................................................................................................................1
1.2 Specifications .............................................................................................................1
1.3 Safety..........................................................................................................................2
Section 2 Description and Assembly of Components...............................................3
2.1 Whole Gel Eluter Components..................................................................................4
2.2 Vacuum Harvester Components................................................................................5
Section 3 Assembly and Operation.............................................................................6
3.1 Preparative Slab Gel Electrophoresis.........................................................................6
3.2 Whole Gel and Mini Whole Gel Eluter Assembly....................................................6
3.3 Running the Whole Gel Eluter.................................................................................10
3.4 Harvesting Eluate.....................................................................................................10
Section 4 Optimizing Conditions ..............................................................................12
4.1 Buffer Selection........................................................................................................12
4.2 Running Conditions .................................................................................................13
4.3 Optimization Procedure ...........................................................................................14
Section 5 Maintenance and Sterilization..................................................................14
5.1 Cleaning the Whole Gel Eluter................................................................................14
5.2 Sterilization of the Whole Gel Eluter.......................................................................14
5.3 Cleaning the Vacuum Harvester..............................................................................14
Section 6 SDS-PAGE..................................................................................................15
6.1 Reagents for SDS-PAGE Slab Gels (Laemmli buffer system)3.............................15
6.2 Preparing SDS-PAGE Gels of any %T ...................................................................16
6.3 Separating Gels - Calculating %T............................................................................17
6.4 Stacking Gel - 4%T (0.125 M Tris pH 6.8).............................................................17
Section 7 Native PAGE...............................................................................................18
7.1 Native PAGE Slab Gels...........................................................................................18
7.2 Reagents for Discontinuous Native PAGE (Ornstein-Davis).................................18
7.3 Prepare Ornstein-Davis Acrylamide Gels ...............................................................19
7.4 Reagents for Continuous Native PAGE ..................................................................19
7.5 Prepare Continuous Gels (10 ml acrylamide monomer solution)...........................21
7.6 Sample Preparation ..................................................................................................21
Section 8 Troubleshooting Guide..............................................................................21
Section 9 Equipment and Accessories......................................................................22
Section 10 References...................................................................................................22
Section 1
General Information
1.1 Introduction
The Whole Gel Eluter provides an alternative method for recovering samples from SDS
and Native PAGE slab gels. It is a preparative electrophoresis instrument which allows simultaneous electro-elution of multiple protein bands separated on a polyacrylamide gel. Elution
is in the transverse direction, through the thickness of the gel. Proteins are eluted into narrow
chambers with each of these fractions containing individual or multiple protein bands.
The eluter consists of a solid acrylic base, a drop-in bottom electrode, an elution chamber core, an upper plate/spring electrode and a lid. The Whole Gel Eluter comes in two sizes.
The large format accommodates slab gels up to 20 cm long and the small format is designed
for mini-gels (Mini-PROTEAN®II and Ready Gels). Filter paper and a cellophane sheet are
placed between the bottom electrode and the elution chamber core. The gel is sandwiched
between the top of the elution chamber core and the spring electrode. During elution, the proteins are captured in one of the 30 elution chambers of the large eluter or one of the 14 chambers of the mini eluter.
One important application for this instrument is the purification and subsequent direct
screening of protein mixtures. Andersen and Heron describe a direct cellular analysis of a
complex protein mixture for biological activity.1The eluter acts as an electrodialyzer removing SDS from protein, leaving them in a non-toxic physiological buffer that can then be used
directly in a cellular assay. The Rotofor®and Prep cell are recommended for larger scale electrophoretic protein purification.
Simple procedures are provided in this manual for optimizing elution conditions. It is
recommended that these procedures be performed for each new sample and when making
changes to slab gel electrophoresis conditions.
1.2 Specifications
Construction
Base acrylic/polycarbonate insert
Elution chamber core acrylic/polycarbonate insert
Bottom electrode platinum coated titanium plate
Upper plate/spring electrode acrylic/platinum coated titanium plate
Lid polycarbonate
Cutting template acrylic
Consumables Large Mini
Upper filter paper 50 sheets 50 sheets
Lower filter paper 75 sheets 50 sheets
Sealing tabs 50 tabs 50 tabs
Cellophane membrane 25 sheets 25 sheets
Other Specifications Large Mini
Shipping weight 9.25 lb 4.75 lb
Overall size 5.0” H x 6.0” W x 6.0” D 5.0” H x 10.5” W x 10.5” D
Voltage limit 300 volts 200 volts
Power limit 15 watts 10 watts
Elution sample volume ~3 ml ~0.5 ml
1
2
1.3 Safety
Power to the Whole Gel Eluter is to be supplied by an external DC power supply. This
power supply must be ground isolated in such a way that its DC voltage output floats with
respect to ground. The recommended power supply for this instrument is the PowerPac 300.
The maximum specified operating parameters are:
Whole Gel Eluter Mini-Whole Gel Eluter
300 VDC 200 VDC
15 W 10 W
The lid assembly provides a safety interlock for the unit. Current flow to the cell is broken when the lid assembly is removed. Do not attempt to circumvent this safety interlock and
always turn off and disconnect the power supply when working with the cell.
The recommended power supplies are ground isolated by design to minimize potential
shock hazard. However, working around high voltage equipment in a laboratory environment
is potentially dangerous. It is the user’s responsibility to always exercise care in setting up
and running electrophoresis instruments. Always turn off the power supply before removing
the lid. Do not attempt to use the cell without the safety lid.
Important:
This Bio-Rad product is designed and certified to meet EN61010-1 safety standards.
Certified products are safe to use when operated in accordance with the instruction manual.
This instrument should not be modified or altered in any way. Alteration of this instrument
will:
• Void the manufacturer’s warranty
• Void the EN61010-1 certification, and
• Create a potential safety hazard
Bio-Rad is not responsible for any injury or damage caused by use of this instrument for
purposes other than those for which it is intended or by modifications of the instrument not
performed by Bio-Rad or an authorized agent.
This product conforms to the class A standards for Electromagnetic Emissions, intended
for laboratory equipment applications. It is possible that emissions from this product may
interfere with some sensitive appliances when placed nearby or on the same circuit as those
appliances. The user should be aware of this potential and take appropriate measures to avoid
interference.
* EN61010-1 is an internationally accepted electrical safety standard for laboratory instruments.
!
Section 2
Description and Assembly of Components
Whole Gel Eluter exploded view.
3
Lid
Upper plate/
spring electrode
Upper chamber
filter paper
Acrylamide gel
Sealing tabs
Elution
chamber core
Cellophane
Lower chamber
filter paper**
Lower plate
electrode
Base
** Mini Whole Gel Eluter is assembled with
2 sheets of lower chamber filter paper.
2.1 Whole Gel Eluter Components
Base and Lower Electrode
The base forms a stable foundation for the eluter. The recess in it houses the lower electrode, the lower filter paper, the cellophane membrane and the elution chamber core.
Adjustable feet enable leveling of the eluter.
Elution Chamber Core
The elution chamber core is a multi-channel collection vessel which is dropped into the
base plate and screwed down over the filter paper and cellophane membrane. Proper sealing
of the core is important for preventing cross-contamination of the fractions. Proteins eluted
from the gel are held in the individual chambers until they are collected with the vacuum harvester or transfer pipet.
Upper Plate/Spring Electrode
Attached to the upper plate is the spring electrode. The electrode makes contact with the
upper filter paper. The tension on the spring holds the slab gel in place and seals the tops of
the elution chambers.
Clamps
The upper plate and base plate are held together by clamps on the sides of the eluter. To
open the clamps of the Whole Gel Eluter, pull the bottom outward as shown in Figure A.
Then disengage the clamp as shown in Figure B. The Mini-Whole Gel Eluter has its clamps
mounted in the opposite direction. To open the clamps of the Mini-Whole Gel Eluter, pull
the top outward. Then disengage the clamp at the bottom. To close the clamps, reverse these
procedures.
A. Step 1
B. Step 2
Opening Whole Gel Eluter Clamps
4
Lid
The lid covers the electrical contacts during elution to provide electrical insulation. The
lid cannot be removed without breaking the electrical circuit. It can be placed on the eluter in
only one alignment, so that the anode and cathode connections cannot be accidentally reversed.
Cutting Template
The cutting template is a guide for excising the portion of the slab gel intended to be eluted. It also functions as a template for cutting your own upper and lower filter paper and cellophane membrane.
Ruler
The ruler has been calibrated to the distance between elution channels. It can be used as
a guide to approximate where individually resolved protein bands may elute.
In order to purify an individual protein, the nearest contaminant must be at least 5 mm
away on a preparative gel for the Mini Whole Gel Eluter or 6 mm for the Whole Gel Eluter.
2.2 Vacuum Harvester Components
* The Mini Whole Gel Eluter uses 14, 1.5 ml microcentrifuge tubes.
5
Vacuum tubing
Harvest needle
array
30, 12 x 75 mm
tubes*
Harvesting lid
Vacuum control
valve
Harvesting
tube rack
Tube Rack
The tube rack forms a stable base for the harvester as well a housing for the collection
tubes. The rack is numbered for easy identification of fractions.
Lid and Needle Array
The lid fits onto the base only one way for proper alignment of the harvester needles with
the collection tubes. Connected to the lid by tubing is the needle array. The needle array inserts
into the harvest ports on the left side of the eluter. Thumb screws hold the needle array in
place, forming a seal between the array and the eluter. The array is designed to be inserted into
the eluter collection ports only one way for proper alignment during harvesting.
Vacuum Control Valve
The vacuum control valve is attached to the lid of the harvesting unit. The valve can be
gradually adjusted to increase or decrease the vacuum preventing uneven harvesting and splattering of eluates.
Section 3
Assembly and Operation
3.1 Preparative Slab Gel Electrophoresis
The Whole Gel Eluters can accommodate SDS or Native PAGE preparative gels up to 3
mm thick. Gel thickness can have a major effect on the elution and recovery efficiency of
proteins. In addition, proteins not eluted into a buffer containing SDS, may adhere to the cellophane (or dialysis) membrane. The thicker the gel, the longer it will take to elute the proteins and the longer they will be in contact with the membrane. Protein recovery can be greatly
improved by reversing the current for 10 to 15 seconds after elution and by keeping elution
times down to a minimum.
1. Run the SDS or Native gel with a preparative comb following standard electrophoresis
procedures. Refer to Sections 6–7 and References for information on running SDS and
Native PAGE gels. The Mini Whole Gel Eluter requires a preparative gel with a minimal
width of 6.5 cm and the large format eluter requires a minimal width of 14.5 cm. The
length of the gel is not critical. It is acceptable for some unused elution chambers to be left
empty. It is convenient to have a lane of prestained standard proteins adjacent to the
preparative well to facilitate alignment of the cutting template, but this lane must be cut
off before placing the gel in the eluter.
2. Make up a liter of elution buffer for the Whole Gel Eluter and 500 ml for the Mini Whole
Gel Eluter. This buffer will be used to equilibrate the preparative slab gel and for the sub-
sequent eluter assembly. Refer to Sections 4.1 and 7.4 for elution buffer selection.
3. Following electrophoresis, remove the gel from the gel cassette and equilibrate it in elu-
tion buffer for 15–20 minutes. This is to allow an exchange of buffers and minimize
swelling of the gels during elution.
3.2 Whole Gel and Mini Whole Gel Eluter Assembly
One of the most important factors in the successful use of the Whole Gel Eluter is the
assembly of the unit. As with blotting techniques, it is vital to remove and (or) prevent bubble formation under the gel. Second, proper alignment of the protein bands parallel to the elution chambers enhances selectivity.
6
Loading...