Bio-Rad 111 Mini IEF Cell User Manual
Model 111 Mini
IEF Cell
Instruction
Manual
Catalog Numbers
170-2975 and 170-2976
For Technical Service
Call Your Local Bio-Rad Office or
in the U.S. Call 1-800-4BIORAD
(1-800-424-6723)
Note
To insure best performance from the Model 111 Mini IEF Cell, become fully acquainted with these
operating instructions before using the cell to separate samples. Bio-Rad recommends that you first read
these instructions carefully. Then assemble and disassemble the cell completely without casting a gel. After
these preliminary steps, you should be ready to cast and run a gel.
Bio-Rad also recommends that all Model 111 Mini IEF Cell components and accessories be cleaned
with a suitable laboratory cleaner (such as Bio-Rad Cleaning Concentrate, catalog number 161-0722) and
rinsed thoroughly with distilled water, before use.
Warranty
Bio-Rad Laboratories warrants the Model 111 Mini IEF Cell against defects in materials and
workmanship for 1 year. If any defects occur in the instrument during this warranty period, Bio-Rad
Laboratories will repair or replace the defective parts free. The following defects, however, are specifically
excluded:
1. Defects caused by improper operation.
2. Repair or modification done by anyone other than Bio-Rad Laboratories or an authorized
agent.
3. Use of fittings or other spare parts supplied by anyone other than Bio-Rad Laboratories.
4. Damage caused by accident or misuse.
5. Damage caused by disaster.
6. Corrosion due to use of improper solvent or sample.
This warranty does not apply to parts listed below:
1. Platinum wire, glass plates.
For any inquiry or request for repair service, contact Bio-Rad Laboratories after confirming the model
and serial number of your instrument.
Model
Catalog No.
Date of Delivery
Serial No.
Invoice No.
Purchase Order No.
Table of Contents
Section 1 General Information................................................................................1
1.1 Introduction ...........................................................................................................1
1.2 Specifications.........................................................................................................1
1.3 Safety.....................................................................................................................2
1.4 Model 111 Mini IEF Cell Components.................................................................3
1.5 Capillary Thin Layer Gel Casting Tray.................................................................3
Section 2 Introduction to Isoelectric Focusing.......................................................4
2.1 The Electrofocusing Principle...............................................................................4
2.2 Carrier Ampholytes...............................................................................................4
2.3 Choice of Support Matrix......................................................................................5
Section 3 Polyacrylamide Gel Isoelectric Focusing...............................................5
3.1 Considerations in Matrix Preparation....................................................................5
3.2 Stock Solutions for Polyacrylamide IEF Gels.......................................................6
3.3 Reagents for Polyacrylamide Electrofocusing Gels..............................................6
3.4 Use of Gel Support Film for Polyacrylamide........................................................7
3.5 Casting Polyacrylamide Gels ................................................................................8
3.6 Sample Preparation................................................................................................9
3.7 Sample Application.............................................................................................10
3.8 Position of Application........................................................................................10
Section 4 Running The Gel....................................................................................10
4.1 Set Up Proceedure...............................................................................................10
4.2 Run Conditions....................................................................................................11
Section 5 Sample Detection and Gel Storage.......................................................12
5.1 Removing the Gel................................................................................................12
5.2 Band Detection....................................................................................................12
5.3 Destaining............................................................................................................13
5.4 Other Detection Methods ....................................................................................13
5.5 Gel Drying and Preservation..............................................................................13
Section 6 Agarose Gel Electrofocusing.................................................................14
6.1 Introduction .........................................................................................................14
6.2 Preparing Agarose Gels.......................................................................................14
6.3 Sample Preparation..............................................................................................16
6.4 Sample Application.............................................................................................16
6.5 Position of Application........................................................................................16
6.6 Set Up Procedure.................................................................................................17
6.7 Run Conditions....................................................................................................17
6.8 Sample Detection.................................................................................................18
Section 7 Troubleshooting .....................................................................................19
7.1 General Troubleshooting.....................................................................................19
7.2 Casting Troubleshooting, Polyacrylamide Gels..................................................20
7.3 Casting Troubleshooting, Agarose Gels..............................................................21
Section 8 References...............................................................................................22
Section 9 Equipment and Accessories..................................................................23
9.1 Equipment............................................................................................................22
9.2 Accessories..........................................................................................................22
9.3 Gel Support Film.................................................................................................22
9.4 Isoelectric Focusing Chemicals and Reagents.....................................................22
9.5 Bio-Lyte®Ampholytes .......................................................................................23
9.6 Stains ...................................................................................................................24
9.7 Sample Preparation..............................................................................................24
9.8 Power Supplies....................................................................................................24
Section 1
General Information
1.1 Introduction
Bio-Rad’s Model 111 Mini IEF Cell introduces a simple and innovative “inverted” gel format
for analytical isoelectric focusing applications. The gel is run upside-down, directly contacting the
electrodes, to eliminate the need for electrode buffers and wicks. There is no need for active
cooling during the focusing process. Condensation within the cell is not a problem because the gel
is run upside-down, and therefore, the condensate cannot fall onto the gel.
The compact Model 111 Mini IEF Cell contains two graphite electrodes with an inter-
electrode distance of 5 cm. The graphite electrodes are removable for easy cleaning. A maximum
applied voltage of 450 volts is sufficient to yield tight bands, with excellent separation, within 90
minutes. The Model 111 Mini IEF Cell makes use of ultra-thin 0.4 mm gels, which provide better
heat dissipation than thicker gels. Casting these gels is simple with the casting tray and gel
support film provided with the cell. Acrylamide and agarose gels are both cast using the same
casting tray.
1.2 Specifications
Construction
Outer chamber Fabricated acrylic
Cell lid Polycarbonate
Casting tray Fabricated acrylic
Electrodes High purity graphite, 0.95 cm diameter
Sample templates Polyvinyl chloride
Shipping weight 1.8 kg
Overall size 24.6 (l) x 11.4 (w) x 4.8 cm (h)
Casting tray 20.3 (l) x 13.9 (w) x 2.3 cm (h)
Gel size 125 (w) x 65 (l) x 0.4 mm (t)
Glass plate size 125 x 65 mm
Voltage/power limit 500 VDC / 5 W
Casting tray temperature limit 60 °C
Note: The Model 111 Mini IEF Cell is not compatible with acetone, chlorinated hydrocarbons
(i.e., chloroform), or aromatic hydrocarbons (i.e., toluene, benzene).
To clean the cell components use a mild detergent, such as Bio-Rad’s Cleaning Concentrate
(catalog number 161-0722) or ethanol.
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1.3 Safety
Power to the Model 111 Mini IEF Cell is to be supplied by an external DC voltage power
supply. This power supply must be ground isolated in such a way that the DC voltage output floats
with respect to ground. All of Bio-Rad's power supplies meet this important safety requirement.
Regardless of which power supply is used, the maximum specified operating parameters for the
cell are:
500VDC maximum voltage limit
5 Watts maximum power limit
50° C maximum ambient temperature limit
Current to the cell, provided from the external power supply, enters the unit through the lid
assembly, providing a safety interlock to the user. Current to the cell is broken when the lid is
removed. Do not attempt to circumvent this safety interlock, and always turn the power supply off
before removing the lid, or when working with the cell in anyway.
IMPORTANT:
This Bio-Rad instrument is designed and certified to meet IEC1010-1* 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 IEC1010-1 safety certification
• Create a potential safety hazard
Bio-Rad is not responsible for any injury or damage caused by the use of this instrument for
purposes other than for which it is intended or by modifications of the instrument not
performed by Bio-Rad or an authorized agent.
*IEC 1010-1 is an internationally accepted electrical safety standard for laboratory instruments.
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!
1.4 Model 111 Mini IEF Cell Components
Fig. 1.1. Model 111 Mini IEF Cell. Outer chamber (1), sliding, interlocking lid with power cables (2), graphite
electrodes (3), casting tray (4), glass plates (5), sample templates (6), gel support film (7), and 5 ml Bio-Lyte
3/10 ampholyte (8).
1.5 Capillary Thin Layer Gel Casting Tray
The Capillary Thin Layer Gel Casting Tray provides the fastest and easiest method for casting
electrofocusing gels.
The casting tray consists of an acrylic plate with precisely defined spacers of 0.4 mm
thickness as shown in Figure 1.2. The acrylic surface imparts a slight inhibitory effect on
acrylamide polymerization, eliminating sticking and tearing of the gel.
Fig. 1.2. Casting tray.
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4
6
7
8
5
2
3
1
Glass plate
Gel
Spacer rails
Section 2
Introduction to Isoelectric Focusing
2.1 The Electrofocusing Principle
Conventional electrophoresis separates proteins and other charged molecules by
electrophoretically-driven migration through a sieving matrix that is buffered at a constant pH.
Each component of the mixture assumes its own characteristic velocity based on molecular size
and surface charge. This velocity is constant throughout the electrophoresis experiment and is
counteracted by diffusion, which tends to broaden the bands. There is no tendency toward
equilibrium in conventional electrophoresis, and the protein bands will run off the gel if the
electrical field is not interrupted.
On the other hand, electrofocusing separates proteins on the basis of surface charge alone as a
function of pH. The separation is done in a non-sieving medium (sucrose density gradient,
agarose, or polyacrylamide gel) in the presence of carrier ampholytes, which establish a pH
gradient increasing from the anode to the cathode. Since a protein contains both positive (amines)
and negative (carboxyl) charge-bearing groups, the net charge of the protein will vary as a
function of pH.
A pH gradient is established concomitantly with protein separation. As the protein migrates
into an acidic region of the gel, it will gain positive charge via protonation of the carboxylic and
amino groups. At some point, the overall positive charge will cause the protein to migrate away
from the anode (+) to a more basic region of the gel. As the protein enters a more basic
environment, it will lose positive charge and gain negative charge, via ammonium and carboxylic
acid group deprotonation, and consequently, will migrate away from the cathode (-). Eventually,
the protein reaches a position in the pH gradient where its net charge is zero (defined as its pI or
isoelectric point). At that point, the electrophoretic mobility is zero. Migration will cease, and a
concentration equilibrium of the focused protein is established.
2.2 Carrier Ampholytes
Carrier ampholytes are complex mixtures of amphoteric buffers that form a smooth pH
gradient in an applied electrical field. During electrofocusing, these buffers stack according to
their individual pIs across the gel, producing a linear gradient. In order for the gradient to appear
smooth and continuous, a large number of these buffering components must be present. This is
also a requirement for separating a complex mixture of proteins.
Bio-Lyte ampholytes are derivatized low-molecular weight amines that are electrophoretically
separated and reblended to give smooth and reproducible gradients. Narrow range Bio-Lyte
ampholytes are produced and tested so that, under normal circumstances, no additional blending
or fortification will be necessary to achieve the desired shallow gradient.
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2.3 Choice of Support Matrix
The electrofocusing process must be stabilized against convection and, to a lesser extent,
diffusion, by a support matrix. This can be anything from a liquid column stabilized by a sucrose
density gradient to a gel cast from agarose or polyacrylamide. The principal criteria for a good
support matrix are that it should be relatively non-sieving so that molecular size is not a factor in
protein mobility, and that it must be free of charged groups which would give rise to internal fluid
flow and distortion of the pH gradient. A complete discussion of electrofocusing matrices is given
in Reference 1.
For analytical work, both agarose and polyacrylamide gels provide good supports for
electrofocusing. Agarose has the advantage of very large pore structures (as large as 500 nm),
making it an ideal non-sieving medium; however, it suffers from varying degrees of residual
negative charge from sulfate groups. For this reason, only agarose proven for electrofocusing
applications should be used (Bio-Rad’s Zero -MrAgarose, catalog number 162-0022). Agarose
concentrations may vary between 0.5 and 1.25%. Proteins as large as 50 x 106daltons have been
successfully electrofocused in agarose.
Section 3
Polyacrylamide Gel Isoelectric Focusing
3.1 Considerations in Matrix Preparation
Because they are prepared from monomers, polyacrylamide gels can be tailored to meet
particular separations requirements. The most common gel composition for horizontal
electrofocusing is T = 5%, C = 3%, where:
g acrylamide + g crosslinker
total solution volume in ml
g crosslinker
g acrylamide + g crosslinker
This formulation will give a suitable non-sieving gel for proteins up to 106daltons, that is still
rigid enough to handle conveniently. A slightly stronger gel of T=5%, C = 4% may be used for
protein samples under 200,000 daltons.
The choice of a catalyst is extremely important in electrofocusing, since any residual ions will
affect the final attainable voltage, and can lead to overheating and gross distortions in the gel. For
this reason a three-phase catalyst system of ammonium persulfate, riboflavin-5'-phosphate, and
TEMED is recommended. This system, catalyzed by light, will give reproducible polymerization
with a minimum of ionic contamination.
The formation of polyacrylamide gels has been extensively studied, and a detailed discussion
of practical considerations is available in Bio-Rad’s bulletin 1156.
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x 100
%C =
x 100
%T =
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