Bio-Rad MicroRotofor Cell User Manual

MicroRotofor

™

Cell

Starter Kit Manual

For Technical Service Call Your Local Bio-Rad Office or in the US Call 1-800-4BIORAD (1-800-424-6723)

Catalog Number

170-2804

Table of Contents

Section 1 Introduction................................................................1

Section 2 Starter Kit Components............................................1

Section 3 Required Equipment and Reagents ........................1

Section 4 Setup and Operation ................................................2

4.1 Overview..............................................................................2

4.2 Prepare the Focusing Assembly..........................................2

4.3 Prepare and Load the Protein Sample ................................4

4.4 Perform the Focusing Run....................................................6

4.5 Power Conditions ................................................................9

4.6 Harvest the Fractions ..........................................................9

Section 5 Disassembly and Cleaning ....................................12

5.1 Focusing Assembly............................................................12

5.2 Harvesting Station..............................................................12

Appendix A Product Information................................................14

Section 1
Introduction

This kit was designed to familiarize you with the MicroRotofor™ Cell before
running your own sample.The setup and operation guides you through the
assembly and a complete fractionation and harvesting of a mixture of three
naturally colored proteins.

Note: For a detailed description of the MicroRotofor™ components, the
setup, and analysis of the results, please refer to the MicroRotofor™
instruction manual.

Section 2
Starter Kit Components

This kit contains:

• Bio-Lyte®Ampholytes.10 ml, pH range 3-10 – Catalog #163-1112

• Protein Sample, 1 ml – Catalog Number 170-2919
Phycocyanin, 2 mg: Blue protein, subunits pI range is 4.5–5.5
Hemoglobin, 2 mg: Red protein, subunits pI range is 6.0–7.5
Cytochrome c, 2 mg: Orange protein, subunits pI range is 8.0–9.0

• Focusing Chamber (1)

• Anode Membranes (Cation Exchange Membranes):2 membranes

equilibrated in 15 ml electrolyte solution 0.1 M H3PO

4

• Cathode Membranes (Anion Exchange Membranes):2 membranes

equilibrated in 15 ml electrolyte solution 0.1 M NaOH

• Harvesting T r ay (1)

• Sample loading syringe, 3 ml (1)

• Electrolyte loading syringe, 10 ml (2)

Section 3
Required Equipment and Reagents

• MicroRotofor™ Cell with Instruction manual

• Power supply capable of 1 Watt constant power control or multistep

constant Voltage control, e.g.the PowerPac™ HV Power Supply
(Catalog N#164-5056)

1

• Vacuum source and vacuum trap.(Vacuum in 22–28 mm Hg range)

• Deionized water

• Pipettes (100 µl – 2.75 ml volumes)

• Beaker or equivalent to hold the 3 ml sample volume.

Section 4
Setup and Operation

Note: For a detailed description of the MicroRotofor™ components, the
setup, and analysis of the results, please refer to the MicroRotofor™
instruction manual.

4.1 Overview

• Prepare the Focusing assembly:Electrode chambers and focusing

chamber with electrode membranes.

• Prepare and load the starter kit protein sample and seal the loading

ports.

• Add electrolyte solutions to the electrode chambers.

• Position the focusing assembly in the chassis and start the IEF run.

• Stop the run, remove the lid, and connect the system to a vacuum

source.

• Remove the loading port sealing tape and position the focusing chamber

into the harvesting station.

• Aspirate the fractions into the harvesting tray.

4.2 Prepare the Focusing Assembly

The focusing assembly consists of the electrode assemblies, the electrode
membranes and the focusing chamber.

2

Fig. 1. MicroRotofor components and accessories. MicroRotofor chassis and lid (1),

harvesting tray (2), focusing chamber (3), anode assemb ly (4a), cathode assemb ly (4b), cathode
membrane, (5a), anode membrane, (5b), 10 ml syringes (6), 3 ml syringe (7), forceps (8),
assembly tool (9), sealing tape (10), cleaning brush (11), vacuum hose (12), vacuum chamber
(13).

1. Rinse the equilibrated ion exchange membranes with deionized water.

2. Insert an anode membrane assembly (red casing) into one end of the

focusing chamber and a cathode membrane assembly (black casing)
into the other end. (Figure 2)

Fig. 2. Inserting an anion exchange membrane into the focusing chamber.

3. Attach the electrode assemblies to the focusing chamber.

a. The anode assembly (red) is attached to the focusing chamber end

containing the anode membrane (red casing).

3

b. The cathode assembly (black) is attached to the focusing chamber

end containing the cathode membrane (black casing).

4. Align one row of focusing chamber ports with the vents on the electrode

assemblies.These are the sample loading ports. (Figure 3)

Fig. 3. Alignment of the sample loading ports with the vents on the anode (left) and
cathode (right) assemblies.

4.3 Prepare and Load the Protein Sample

Prepare the protein sample

The protein sample included in the starter kit contains three naturally colored
proteins in deionized water, Phycocyanin (pI range 4.5–5.5), Hemoglobin
(pI range 6.0–7.5), and Cytochrome c (pI range 8.0–9.0).Each protein is
present at a concentration of 2 mg/ml for a total protein concentration of
6 mg/ml.

Note: Vortex sample vial to homogenize material.

• To 100 µl of protein sample (600 µg total protein), add 150 µl Bio-lytes

pH 3-10, and 2.75 ml deionized water for a total volume of 3.0 ml.

Load the Protein Sample

The focusing chamber features two rows of ports.The row that is aligned
with the vents on the electrode assemblies will be used to load the sample
(loading ports).The row on the opposite side of the focusing chamber will be
used for harvesting the fractions (harvesting ports).The harvesting ports
must be sealed with tape before the sample is loaded.After the sample is
loaded, the loading ports will also be sealed with a piece of tape.

4