Preface |
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vii | |
Note to Instructors |
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viii | |
Acknowledgments |
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viii | |
INTRODUCTORY UNIT |
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Introductory Lecture Introduction to the Biochemical Laboratory |
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1 | (49) |
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Theory/Principles Course Description |
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6 | (2) |
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Theory/Principles ``Central Dogma of Molecular Biology'' |
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8 | (1) |
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Theory/Principles Laboratory Safety |
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9 | (2) |
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Theory/Principles The Scientific Method: Surviving Recipe Mentality |
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11 | (2) |
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Theory/Principles Proactive Troobleshooting |
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13 | (3) |
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Theory/Principles Introduction to the Biotechnology Laboratory |
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16 | (4) |
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Theory/Principles Error Analysis and Assay Sensitivity |
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20 | (2) |
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Theory/Principles Treatment of Analytical Data |
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22 | (8) |
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Theory/Principles Concentration and Temperature Effects on pKa |
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30 | (5) |
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Introductory Lab 1 Basic Biochemical Techniques I: Pipet Calibration and Solution Preparation |
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35 | (3) |
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38 | (1) |
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Introductory Lab 2 Basic Techniques II: Absorbance Spectroscopy and Protein Concentration Determinations |
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39 | (4) |
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Process Rationale AMP and Tryptophan Absorbance Spectra; Sample Calculations |
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43 | (1) |
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Theory/Principles Absorption Data for the Nucleoside Monophosphates |
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44 | (2) |
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Process Rationale Absorption Spectra Data for the Aromatic Amino Acids at pH 6; UV Absorption Characteristics of the Aromatic Amino Acids. Selected Extinction Coefficients |
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46 | (1) |
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Process Rationale BCA Assay Sample Data |
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47 | (1) |
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Innovation/Insight Measurement of Protein in 20 Seconds |
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48 | (2) |
Part 1 NUCLEIC ACIDS AND CLONING |
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50 | (1) |
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51 | (1) |
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52 | (27) |
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Theory/Principles Subcloning Procedure |
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60 | (1) |
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Innovation/Insight The pET Bacterial Plasmid System (Novagen) |
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61 | (3) |
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Media Preparation; Bacterial Growths; Plasmid Minipreps; HindIII Digestion of DNA, Commercial Bacteriophage λ DNA BstEII Digest Size Standards |
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64 | (9) |
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Process Rationale pUR278 and p2D Restriction Maps |
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67 | (1) |
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Process Rationale Cloning the myo-3 Gene from C. elegans and Construction of an Expression Vector |
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68 | (1) |
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Process Rationale C. elegans myo-3 Gene in pUR288 |
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69 | (1) |
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Vendor Literature Restriction Enzymes HindIII and BstEII; λ DNA Digests |
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70 | (2) |
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Process Rationale Phage λ BstEII Digest |
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72 | (1) |
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Agarose Gel Electrophoresis; Photography of HindIII Plasmid Digests Visualized by Fluorescence of Intercalated Ethidium |
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73 | (6) |
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Exercises Restriction Mapping |
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76 | (3) |
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Construction of Recombinant Plasmids |
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79 | (28) |
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Innovation/Insight Protecting and Manipulating Large DNA Substrates |
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82 | (1) |
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Innovation/Insight Yeast of Burden - Yoking the YAC |
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83 | (4) |
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Extraction and Cleanup of DNA Bands Cut from Agarose Gels, Quantitation of Yields, and Ligation of myo-3 HindIII DNA Insert Fragment into Linearized β-gal Plasmid DNA |
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87 | (20) |
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Vendor Literature Gibco BRL™ T4 DNA Ligase |
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93 | (6) |
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Vendor Literature DNA Purification Kit (NaI/Glass Bead Method) |
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99 | (6) |
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Alternative Approach The Use of β-Agarase to Recover DNA from Gel Slices |
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105 | (1) |
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Alternative Approach GELase™ |
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106 | (1) |
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The Polymerase Chain Reaction |
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107 | (18) |
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Innovation/Insight Polymerase Chain Reaction Used for Antigen Detection |
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117 | (1) |
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Immuno-PCR: Very Sensitive Antigen Detection by Means of Specific Antibody-DNA Conjugates |
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117 | (4) |
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Polymerase Chain Reaction Test for myo-3 Gene Insert Orientation |
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121 | (4) |
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Transcription of Genomic DNA and Analysis of the Resulting mRNAs |
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125 | (12) |
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Alternative Approach Isolation of Total RNA from E. coli Cells |
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128 | (3) |
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Alternative Approach Promega™ PolyATract™ System 1000 |
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131 | (5) |
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Alternative Approach Electrophoresis and Northern Blotting of RNA |
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136 | (1) |
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Transformation and Gene Expression |
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137 | (28) |
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Innovation/Insight How Cells Respond to Stress |
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140 | (8) |
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Preparation of Fresh Transformation-Competent Cells |
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148 | (4) |
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Alternative Approach Ultracomp™ Transformation Kit |
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150 | (2) |
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Colony Immunoblotting to Screen for Transformants |
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152 | (13) |
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Alternative Approach The QIA expressionist, QIAGEN™ |
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155 | (10) |
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Analysis of DNA or RNA by Duplex Hybridization: DNA Isolation, Labeling, and Probing |
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165 | (33) |
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Innovation/Insight Reduction of Background Problems in Nonradioactive Northern and Southern Blot Analyses Enables Higher Sensitivity than 32P-Based Hybridization |
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168 | (7) |
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Labeling of DNA and Probe Construction from Cloned C. elegans myo-3 Gene; Quantitation of DNA Concentration |
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175 | (15) |
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Vendor Literature Digoxigenin Labeling of DNA: Genius™ Nucleic Acid Labeling System |
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177 | (13) |
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Isolation of C. elegans Genomic DNA, Quantitation of DNA Concentration, and Digestion to Extract the myo-3 Gene |
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190 | (2) |
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192 | (4) |
Part 2 PROTEIN PURIFICATION |
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196 | (1) |
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197 | (1) |
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198 | (123) |
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Theory/Principles Preparation and Handling of Biological Macromolecules for Crystallization |
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204 | (9) |
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Theory/Principles Solution Structure of Biomacromolecules in Ionic Solutions |
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213 | (6) |
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Theory/Principles Solubility as a Function of Protein Structure and Solvent Components |
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219 | (12) |
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Theory/Principles Dominant Forces in Protein Folding |
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231 | (30) |
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Alternative Approach Hydrophobic Interaction Chromatography |
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261 | (5) |
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Alternative Approach Centriprep Microconcentrators for Small Volume Concentration; Centricon-3 and Centricon-100 |
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266 | (5) |
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Innovation/Insight Subcellular Fractionation |
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271 | (2) |
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The Protein Purifier: A Learning Aid from Pharmacia |
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273 | (5) |
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Induction and Purification of β-Galactosidase Fusion Protein from Bacteria |
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278 | (2) |
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Gel Filtration of Molecular Weight Standards and Protein Fractionation |
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280 | (10) |
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Process Rationale Gel Filtration Chromatography |
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282 | (4) |
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Vendor Literature Sephadex and Sephacryl |
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286 | (2) |
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Vendor Literature Sigma™ Gel Filtration Molecular Weight Markers |
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288 | (2) |
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Microplate β-Galactosidase Assay to Determine Fractions Containing Fusion Protein; MW Determination |
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290 | (6) |
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Process Rationale Time Course Assay of β-Galactosidase |
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292 | (2) |
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Vendor Literature β-Galactosidase Substrates |
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294 | (1) |
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Innovation/Insight Luminescent Reporter Gene Assays for Luciferase and β-Galactosidase Using a Liquid Scintillation Counter |
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295 | (1) |
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Ion Exchange Column Chromatography |
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296 | (11) |
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Process Rationale Ion Exchange Chromatography |
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298 | (4) |
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Theory/Principles The Isoelectric Point: Protein Charge Neutrality at a Particular pH |
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302 | (1) |
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Alternative Approach Ion-Pair Chromatography |
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303 | (2) |
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Alternative Approach HPLC: Ion Exchange and Reverse Phase Methods: Literature Sources |
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305 | (2) |
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Affinity Chromatography and Microplate β-Galactosidase Assays to Determine Fractions Containing Fusion Protein |
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307 | (10) |
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Process Rationale Affinity Chromatography |
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310 | (4) |
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Process Rationale Affinity Chromatography: One Step Purification of Hybrid Proteins Carrying Fused β-Galactosidase Activity |
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314 | (3) |
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BCA Protein Concentration Assays and β-Galactosidase Assays to Construct an Enzyme Purification Table |
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317 | (4) |
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Discontinuous Gel Electrophoresis, Protein Mobilities, and Apparent Size Determination |
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321 | (8) |
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Process Rationale Discontinuous Gel Electrophoresis and Protein Size Determination |
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323 | (3) |
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Discontinuous SDS Gel Electrophoresis |
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326 | (3) |
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Immunochemical Techniques |
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329 | (40) |
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Innovation/Insight Immunochemical Techniques |
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331 | (7) |
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Innovation/Insight: The Enzyme Linked Immunosorbent Assay (ELISA) |
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338 | (10) |
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Innovation/Insight How the Immune System Learns About Self |
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348 | (7) |
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Innovation/Insight Making Monoclonal Antibodies That Won't Fight Back |
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355 | (6) |
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361 | (8) |
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Process Rationale Immunoblotting |
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365 | (1) |
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Process Rationale Western Blots Using Stained Protein Gels |
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366 | (3) |
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Combinatorial Biochemical Technology |
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369 | (18) |
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Innovation/Insight Examples of Combinatorial Techniques |
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375 | (1) |
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Innovation/Insight Making Antibody Fragments Using Phage Display Libraries |
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376 | (3) |
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Innovation/Insight Building a Better Enzyme |
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379 | (5) |
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Innovation/Insight The ImmunoZAP™ Cloning and Expression System |
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384 | (3) |
APPENDICES |
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387 | (36) |
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388 | (4) |
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392 | (105) |
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497 | |
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403 | (7) |
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Literature Sources for Biochemical Analyses, Methods, and Preparations |
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410 | (2) |
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Copyright Acknowledgments |
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412 | (4) |
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416 | (2) |
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Suggested Instructions for Lab Reports |
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418 | (1) |
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419 | (4) |
Index |
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423 | |