Bio-Rad Rotofor® and Mini Rotofor Cells - Manual (Page 2)

Table of Contents
Page
Section 1 General Information ......................................................................1
1.1 Introduction................................................................................................1
1.2 Specifications ............................................................................................2
1.3 Isoelectric
Focusing ...................................................................................3
1.4 Safety ........................................................................................................4
Section 2 Description of Major Components ...............................................5
Section 3 Setting Up For A Run ....................................................................6
3.1 Equilibration of the Ion Exchange Membranes ...........................................6
3.2 Assemble the Electrodes ...........................................................................7
3.3 Assemble the Focusing Chamber ..............................................................9
3.4 Prepare the Focusing Chamber ...............................................................10
3.5 Load the Sample......................................................................................10
3.6 Seal the Loading Ports.............................................................................10
3.7 Remove Air Bubbles ................................................................................11
Section 4 Running Conditions ....................................................................11
4.1 Starting the Fractionation .........................................................................11
4.2 Power
Supply ..........................................................................................12
4.3 Fraction Collection ...................................................................................13
4.4 Refractionation.........................................................................................13
4.5 Final
Purification ......................................................................................14
Section 5 Disassembly and Cleaning.........................................................14
Section 6 Sample Preparation.....................................................................15
6.1 Salt Concentration ...................................................................................15
6.2 Clarification..............................................................................................15
6.3 Solubility ..................................................................................................15
Section 7 Optimizing Fractionation ............................................................16
7.1 Ampholyte Choice....................................................................................16
7.2 Sample Capacity......................................................................................17
7.3 Power Conditions.....................................................................................17
7.4 Cooling ....................................................................................................17
7.5 Electrolytes ..............................................................................................18
7.6 Pre-running the Cell .................................................................................18
7.7 Prefocusing..............................................................................................18
7.8 Refractionation.........................................................................................19
Section 8 Analysis of Results .....................................................................19
8.1 Fraction Analysis .....................................................................................19
8.2 Separation of Ampholytes From Proteins.................................................19
Section 9 Troubleshooting Guide ...............................................................20
9.1 Solubility and Precipitation of Proteins .....................................................20
9.2 Factors Affecting the pH Gradient ............................................................21
9.3 Recovery of Biological Activity .................................................................22
9.4 Maximizing
Resolution .............................................................................23
9.5 Power Related Conditions........................................................................24
9.6 Uneven
Harvesting ..................................................................................25
9.7 Mechanical Problems...............................................................................25