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.

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.

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.

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.

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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