Experiments in Biochemistry A Hands-On

Make the most of your lab time with this interactive manual by text author Shawn O. Farrell and co-author Lynn E. Taylor. Youall find a selection of classroom-tested experiments designed to be completed during a normal laboratory period.

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 Concentrations from Graphs

66

(2)

3.7 Tricks of the Trade: Choosing Test Tubes and Pipets

68

(2)

Experiment 3 Beer's Law and Standard Curves

70

(7)

Experiment 3a Protein Concentration of LDH Fractions

77

(6)

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 version)

95

(8)

Experiment 4a Purification of LDH (comprehensive version)

103

(8)

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 from Columns

121

(2)

5.6 Thin Layer Chromatography

123

(1)

5.7 Why Is This Important?

124

(1)

Experiment 5 Separation and Identification of Amino Acids

125

(8)

Experiment 5a Purification of LDH with Ion Exchange Chromatography

133

(10)

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 Chromatography of LDH

169

(10)

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 Isozymes (short version)

218

(5)

Experiment 9a Native Gel Separation of LDH Isozymes (comprehensive)

223

(6)

Experiment 9b SDS-Polyacrylamide Gel Electrophoresis (short version)

229

(6)

Experiment 9c SDS-Polyacrylamide Gel Electrophoresis (comprehensive)

235

(8)

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 Proteins

247

(6)

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 Fragments

269

(8)

Chapter 12 Cloning and Expression of Foreign Proteins

277

(34)

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 Barracuda LDH-A

292

(19)

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

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Experiments in Biochemistry A Hands-On Approach

Summary

Table of Contents

Supplemental Materials