Contents
Preface .............................................................xi
Chapter 1: Basic Practices and Techniques in the Biochemistry Laboratory 1
Laboratory Safety ....................... .1
Laboratory Safety Equipment .........................3
Safe Use of Laboratory Reagents ........................4
Philosophy and Design of Experiments ........................5
Ethics in Science ...................... ..10
Presentation and Analysis of Data ...................... 13
Basic Procedures ...................... 16
Various Instruments Used ...................... 23
Other General Techniques ........... ... ...29
Sample Calculations ... ... 34
Buffers and pH ... ... ...36
Keeping a Laboratory Notebook ... ... ....44
Laboratory Reports ... ... 46
Appendix 1-1: Calculating Titration Curves for Polyprotic Acids and other Multiple
Binding Site Receptors ... ... ... .49
Equipment Used in this Book ... ... ...51
Chapter 2: Spectrophotometry ................................................................ ....53
Introduction ... ... ....53
Design and Properties of Spectrophotometers ... ... .57
Effects of Spectral Bandpass and Stray Light ... ... ..60
Split Beam Recording Spectrophotometers ... ... .62
Chromogenic Reactions Used for Analysis ... ... ..62
Experiment 2-1 to 2-4: ... ... ..70
Reagents Needed for Chapter 2 ... ... . 75
Chapter 3: Quantification of Protein Concentration ......................................................77
Puposes of Protein Quantification ... ... ..77
Common Procedures for Protein Quantification ... ... ....78
Colorimetric Procedures for Quantification of Proteins ... ... ...79
Experiment 3-1: Determination of Protein Concentration ... ... 84
Reagents Needed for Chapter 3 ... ... ...88
Chapter 4: Chromatography .................................................... ..89
Introduction ... ... .89
Gel-Filtration (Size Exclusion or Gel Permeation) Chromatography ... ... ..93
Affinity Chromatography ... ... 100
Ion-Exchange Chromatography ... ... ..108
Hydrophobic-Interaction Chromatography ... ... .113
Experiments 4.1- 4-2 ... ... 117
Reagents Needed for Chapter 4 ... ... .124
Chapter 5: Gel Electrophoresis of Proteins .............................................................125
Process of Electrophoresis ... ... .125
Polyacrylamide Gels ... ... .127
SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) of Proteins....... ... ......... 129
Detection of Proteins in SDS-Polyacrylamide Gels............................. ... ............. 135
Applications of SDS-PAGE ............................................................. ... ..............136
Simple (Nondenaturing, Native) Gel Electrophoresis ......................... ... ..............140
Experiments 5-1 to 5-2 .................................................................... ... ..............147
Chapter 6: Overview of Protein Purification .......................................................157
Introduction ................................................................... ... .................................157
Why Do You Want This Protein? ...................................... ... .............................158
Development of a Suitable Assay Procedure ........................ ... ...........................159
Time, Temperature, and Yield ........................................... ... ..............................161
Selection of the Best Source Material ................................. ... .............................161
Solubilization of the Protein ...................................................... ... .......................163
Initial Steps of Purification .............................................................. ... ................165
Developing a Series of High-resolution Chromatographic Steps ................. ... .....167
Methods Used to Change Buffer and Concentrate Protein Samples . ... ...............169
A Logical Series of Steps .................................................................. ... ............170
Storage of the Purified Protein ........................................................... ... ............170
The Protein Purification Table ................................................................. ... .......171
Chapter 7: Isolation and Characterization of The Enzyme alkaline phosphatase from Escherichia Coli .................................................................................................175
Objectives .............................................................................................. ... .........175
Introduction and Basic Principles ........................................................... ... ...........175
Experiment 7-1 ................................................................................... ... .............175
Characterization of Purified Alkaline Phosphatase ......................................... ... ......190
Appendix 7-1 Assay of Alkaline Phosphatase ............................................... ... ......191
Reagents and Equipment Needed for Chapter 7......................... ... ......................... 196
Chapter 8: Enzyme Kinetics .......................................................................................199
Why Use Steady-State Kinetics ........................................................ ... ..................199
Steady-State Kinetics Principles .......................................................... ... ..............200
Significance of Km and Vmax .............................................................. ... ...............202
Graphical Analysis .............................................................................. ... ..............204
Competitive, "Noncompetitive," and "Uncompetitive" Inhibitors......................... ... ...... 206
Experiments 8.1 to 8-2 ............................................................................. ... .........214
Reagents Needed for Chapter 8 ................................................................ ... .........218
Chapter 9: Enzymatic Methods of Analysis ...............................................................219
Enzymatic Analysis of substrates ........................................................... ... .........219
Assays for Enzymatic Activity.............................................................. ... ........... 220
Practical Considerations ....................................................................... ... ..........221
Coupled Assays .................................................................................. ... ..........222
Experiments with Pyridine Nucleotide-Requiring Enzymes ..................... ... ...........223
Experiment 9-1 to 9-4 ............................................................................ ... ........227
Reagents Needed for Chapter 9............................................................ ... ......... 239
Appendix 9-1: Sample Calculations ..................................................... ... ..........242
Chapter 10: Ligand Binding ...................................................................................247
Ligand Binding is the Key to Most Biological Processes..................................... ......... 247
Analysis of Ligand Binding at Equilibrium ........................................... ... ..........249
Digression on Regression ..................................................................... ... ......252
Effects of the Concentration of L and R ..................................... ..........................255
Effects of Two Sites or Cooperativity ................................................. .................256
Analysis of the Kinetics of Ligand Binding............................................ .............. 261
Methods Used to Study Receptor-Ligand Interactions ..................... ....................264
Experiment 10-1........................................................................... .................. 272
Reagents Needed for Chapter 10 ................................................... ..................275
Chapter 11: Recombinant DNA Techniques ......................................................277
Introduction ................................................................................. ..................277
Overview of Strategy of Recombinant DNA Techniques ....................... ..............278
Cutting and Splicing DNA ............................................................... ...............280
Gel Electrophoresis of DNA .......................................................... .................283
Introducing DNA into Cells ................................................................ ............286
Identifying Transformed Cells ..................................................... ....................287
There Are Many Useful Tool for Recombinant DNA Experiments ....... ...............291
Applications of Recombinant DNA Technology ........................... ....................295
Experiments 11-1 to 11-4 .............................................................. ..............300
Reagents Needed for Chapter 11 .................................................... .............310
Chapter 12: Polymerase Chain Reaction (PCR) Technology .........................313
Introduction ..................................................................... ..........................313
Principle of the PCR Method ........................................... ............................313
Applications of PCR................................................................. .................. 316
Experiments 12-1 to 12-5 ........................................................... ................324
Materials and Reagents Needed for Chapter 12.............................. .............. 335
Chapter 13: Using the Personal Computer and the Internet for Biochemical Research........................................................................................................ 337
Introduction to the World Wide Web (the Internet) .......... ..............................337
Information on the Internet useful to Biochemists .................. .......................344
Using RasMol to View Molecular Structures ............................. ...................352
Other Useful Information on the Internet ...................................... ................354
Homework Assignment ................................................................. ............356
Materials needed for Chapter 13 ....................................... .........................360
Appendix 1: Safety Checklist......................................................................... 361
Appendix 2: an example of an MSDS ............................................................362
Index .................................................................................................................365