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