Handbook of detection of enzymes on electrophoretic gels / 2nd ed.



Part I Introduction
References for Part I....................................................................................3


Part II General Principles of Enzyme Detection on Electrophoretic Gels ........................................................5

Section 1 Chromogenic Reactions.........................................................................5
Products Reducing Tetrazolium Salts.........................................................5
Products Capable of Coupling with Diazonium Salts...........................................6
Products that Cause a pH Change.............................................................7
Orthophosphate..............................................................................8 8
Pyrophosphate...............................................................................9
Hydrogen Peroxide. ................................................................. 9
Carbonate Ions............................................................................ 10
Colored Products ..........................................................................10
Products Bearing Reduced Thiol Groups .....................................................10
Products that Influence the Starch-Iodine Reaction........................................ 11
Products of Polymerizing Enzymes ......................................................... 11
Products of Depolymerizing Enzymes........................................................ 11

Section 2 Fluorogenic Reactions ...................................................................... 12
NADH and NADPH ................................................................... 12
4-Methylumbelliferone......................................................................12
Products that Form Luminescent Lanthanide Chelates ....................................... 13
Miscellany................................................................................ 13

Section 3 Autoradiography ............................................................................ 13

Section 4 Bioautography .............................................................................. 14

Section 5 Two-Dimensional Gel Spectroscopy............................................................ 14

Section 6 Immunoblotting.............................................................................. 15
Immobilization Matrices................................................................... 15
Transfer of Proteins ..................................................................... 16
Specific Antibodies and Labeled Anti-Antibodies .......................................... 17
Detection of Transferred Proteins on Blots ................................................17

Section 7 Miscellanies................................................................................ 18
Detection by Incorporating Water-Insoluble Substrates into Separating Gels................ 18
Detection of Lipid-Metabolizing Enzymes .................................................. 18
Detection by a Two-Dimensional Electrophoresis Procedure ................................. 18
Detection of Enzymatic Proteins by Specific Probes........................................ 19
Labeled Aptamers and Aptamer Beacons as Specific Probes ............................ 19
Labeled Inhibitors as Specific Probes ...............................................19
Specific Detection of Some Nonenzymatic Proteins ......................................... 20
Detection of Proteinase and Ribonuclease Inhibitory Proteins Using Reverse Zymography ............20
Detection of Proteinase Inhibitory Proteins Using Biotinylated Proteinases as Specific Probes .....20




Biotinylated Hyaluronan as a Specific Probe for Hyaluronan-Binding Proteins ..........20
Transferrin...........................................................................20
References for Part II.....................................................................20


Part III Methods of Detection of Specific Enzymes ................................................................23

Section 1 The Structure of Enzyme Sheets ...............................................................23
References ............................................................................... 25

Section 2 General Considerations, Comments, and Recommendations.........................................25
The Choice of Support Medium for Enzyme Electrophoresis and Detection .....................25
Cellulose Acetate Gel.................................................................25
Polyacrylamide Gel....................................................................26
Starch Gel............................................................................27
Strategies of Gel Staining................................................................ 27
The Choice of Zymogram Method.........................................................28
Preparation of Staining Solution......................................................28
Modes of Application of Staining Solutions........................................... 29
Modes of Enhancement of Staining Intensity of Enzyme Activity Bands...................30
Specificity of Zymogram Methods and Some Related Problems.............................31
Recording and Preservation of Zymograms....................................................33
Resource-Saving Strategies.................................................................34
Simultaneous Detection of Several Enzymes on the Same Gel ............................34
Successive Detection of Different Enzymes on the Same Gel.............................35
The Reuse of Staining Solutions ......................................................36
The Use of Several Origins on One Gel ................................................36
Multiple Replication of Electrophoretic Gels by Electroblotting.......................36
The Use of Semipreparative Gels for Production of Preparations of Linking Enzymes ....36
Troubleshooting ............................................................. 37
Safety Regulations ....................................................................... 37
References.................................................................................38

Section 3 Enzyme Sheets*...............................................................................41

Appendix A-1 Minimal Medium for Escherichiacoli....................................................541

Appendix A-2 Citrate Medium for Pediococcus cerevisiae............................................542

Appendix B Alphabetical List of Enzymes..........................................................544

Appendix C Buffer Systems Used for Enzyme Electrophoresis........................................549