Introduction to the Text iii
Objectives of the Biochemistry Laboratory xv (1)
Chapter Format of Experiments in Biochemistry xvi
Chapter 1 Biochemistry Boot Camp 1 (36)
1.1 Lab Safety 2 (1)
1.2 Scientific Notation 3 (1)
1.3 Significant Figures 4 (1)
1.4 Statistics and Scientific Measurements 5 (3)
1.5 Units 8 (1)
1.6 Concentration of Solutions 9 (2)
1.7 Dilutions 11 (1)
1.8 Graphing 12 (11)
1.9 Pipets and Pipetmen(R) 23 (6)
Experiment 1 Use of Pipettors 29 (8)
Chapter 2 Acids, Bases, and Buffers 37 (22)
2.1 Strong Acids and Bases 38 (1)
2.2 Weak Acids and Bases 39 (1)
2.3 Polyprotic Acids 40 (1)
2.4 Buffers 41 (2)
2.5 Good's Buffers 43 (1)
2.6 Choosing a Buffer 44 (2)
2.7 Effect of Concentration and Temperature 46 (1)
2.8 How We Make Buffers 47 (1)
2.9 The Big Summary 47 (2)
2.10 Why Is This Important? 49 (1)
2.11 Expanding the Topic 49 (2)
Experiment 2 Preparation of Buffers 51 (8)
Chapter 3 Spectrophotometry 59 (24)
3.1 Absorption of Light 59 (2)
3.2 Beer-Lambert Law 61 (2)
3.3 Standard Curves 63 (2)
3.4 Protein Assays 65 (1)
3.5 Why Is This Important? 66 (1)
3.6 Expanding the Topic: Calculating 66 (2)
Concentrations from Graphs
3.7 Tricks of the Trade: Choosing Test 68 (2)
Tubes and Pipets
Experiment 3 Beer's Law and Standard Curves 70 (7)
Experiment 3a Protein Concentration of LDH 77 (6)
Fractions
Chapter 4 Enzyme Purification 83 (28)
4.1 Enzymes as Catalysts 83 (2)
4.2 Enzyme Purification 85 (3)
4.3 Units of Enzyme Activity 88 (2)
4.4 Calculating Initial Velocity 90 (2)
4.5 Purification Tables 92 (1)
4.6 Assay for Lactate Dehydrogenase (LDH) 93 (1)
4.7 Why Is This Important? 93 (1)
4.8 Tricks of the Trade 94 (1)
Experiment 4 Purification of LDH (short 95 (8)
version)
Experiment 4a Purification of LDH 103(8)
(comprehensive version)
Chapter 5 Ion Exchange Chromatography 111(32)
5.1 Amino Acids as Weak Acids and Bases 111(2)
5.2 Isoelectric Point 113(5)
5.3 Ion Exchange Chromatography 118(2)
5.4 Ion Exchange Resins 120(1)
5.5 Identification of Compounds Eluted 121(2)
from Columns
5.6 Thin Layer Chromatography 123(1)
5.7 Why Is This Important? 124(1)
Experiment 5 Separation and Identification 125(8)
of Amino Acids
Experiment 5a Purification of LDH with Ion 133(10)
Exchange Chromatography
Chapter 6 Affinity Chromatography 143(14)
6.1 Affinity Chromatography 143(1)
6.2 Gel Supports 144(1)
6.3 Affinity Ligands 145(2)
6.4 Elution of Bound Molecules 147(1)
6.5 Why Is This Important? 147(1)
Experiment 6 Affinity Chromatography of LDH 148(9)
Chapter 7 Gel Filtration Chromatography 157(22)
7.1 Gel Filtration 157(1)
7.2 Types of Supports 158(1)
7.3 Determining the Molecular Weight 159(1)
7.4 Distribution Coefficients 160(2)
7.5 Why Is This Important? 162(1)
7.6 Expanding the Topic 163(2)
Experiment 7 Gel Filtration Chromatography 165(4)
Experiment 7a Gel Filtration 169(10)
Chromatography of LDH
Chapter 8 Enzyme Kinetics 179(28)
8.1 Reaction Rates 179(1)
8.2 Order of Reactions 180(1)
8.3 Michaelis-Menten Approach 180(3)
8.4 Significance of K(m) and V(max) 183(1)
8.5 Linear Plots 184(3)
8.6 Properties of Tyrosinase 187(1)
8.7 Why Is This Important? 187(2)
Experiment 8 Enzyme Kinetics of Tyrosinase 189(10)
Experiment 8a Enzyme Kinetics of LDH 199(8)
Chapter 9 Electrophoresis 207(36)
9.1 Electrophoresis 207(1)
9.2 Agarose Gels 208(3)
9.3 Polyacrylamide Gels 211(2)
9.4 SDS-PAGE 213(2)
9.5 Staining Gels 215(1)
9.6 Lactate Dehydrogenase 216(1)
9.7 Why Is this Important? 216(1)
9.8 Expanding the Topic 217(1)
Experiment 9 Native Gel Separation of LDH 218(5)
Isozymes (short version)
Experiment 9a Native Gel Separation of LDH 223(6)
Isozymes (comprehensive)
Experiment 9b SDS-Polyacrylamide Gel 229(6)
Electrophoresis (short version)
Experiment 9c SDS-Polyacrylamide Gel 235(8)
Electrophoresis (comprehensive)
Chapter 10 Western Blots 243(18)
10.1 Western Blot Theory 243(1)
10.2 Antibodies 244(1)
10.3 Color Development 245(1)
10.4 Blocking and Washing 246(1)
10.5 Why Is This Important? 246(1)
Experiment 10 Western Blots of Serum 247(6)
Proteins
Experiment 10a Western Blot of LDH 253(8)
Chapter 11 Restriction Enzymes 261(16)
11.1 Restriction Nucleases 261(1)
11.2 Restriction Maps 262(2)
11.3 Agarose Gel Separation of DNA 264(1)
11.4 Staining DNA 265(1)
11.5 Phage (Lambda) DNA 266(2)
11.6 Why Is This Important? 268(1)
Experiment 11 Analysis of DNA Restriction 269(8)
Fragments
Chapter 12 Cloning and Expression of Foreign 277(34)
Proteins
12.1 Recombinant DNA 277(2)
12.2 Vectors 279(1)
12.3 Foreign DNA 280(1)
12.4 Restriction Enzymes 281(4)
12.5 Cell Lines 285(1)
12.6 Transformation 286(1)
12.7 Selection 287(1)
12.8 Expression 288(2)
12.9 Why Is This Important? 290(2)
Experiment 12 Cloning and Expression of 292(19)
Barracuda LDH-A
Chapter 13 Polymerase Chain Reaction 311
13.1 Amplification of DNA 311(1)
13.2 Taq Polymerase 312(1)
13.3 Primers 313(3)
13.4 Changing Restriction Sites 316(1)
13.5 Why Is This Important? 317(1)
Experiment 13 PCR of Barracuda LDH-A 318