Fundamental laboratory approaches for biochemistry and biotechnology:
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| Main Authors: | , , |
|---|---|
| Format: | Book |
| Language: | English |
| Published: |
Hoboken, NJ
Wiley
2010
|
| Edition: | 2. ed. |
| Subjects: | |
| Online Access: | Inhaltstext Inhaltsverzeichnis |
| Physical Description: | XIX, 458 S. Ill., graph. Darst. |
| ISBN: | 9780470087664 |
Staff View
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| 100 | 1 | |a Ninfa, Alexander J. |e Verfasser |4 aut | |
| 245 | 1 | 0 | |a Fundamental laboratory approaches for biochemistry and biotechnology |c Alexander J. Ninfa ; David P. Ballou ; Marilee Benore |
| 250 | |a 2. ed. | ||
| 264 | 1 | |a Hoboken, NJ |b Wiley |c 2010 | |
| 300 | |a XIX, 458 S. |b Ill., graph. Darst. | ||
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Record in the Search Index
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| adam_text |
Contents
PREFACE
xv
CHAPTER
1 :
GETTING STARTED IN SCIENTIFIC RESEARCH
1
1.1
The Difference Between Experiments and Demonstrations
.1
1.2
Philosophy and Design of Experiments
.3
1.3
Designing Informative Experiments
.4
1.4
Ethics in Science
.5
1.5
Keeping a Laboratory Notebook
.9
1.6
Laboratory Reports
.13
1.7
Presentation and Analysis of Data
.15
1.8
The
Minisymposium
.18
CHAPTER
2:
BASIC PROCEDURES IN THE
BIOCHEMISTRY LABORATORY
21
2.1
Laboratory Safety
.21
2.2
Special Safety Procedures Are Required for Using Radioactive
Materials or Operating the Autoclave
.26
2.3
Measurement of Weights, Volumes, and
pH.30
2.4
Various Instruments Used
.38
2.5
Other General Techniques
.45
2.6
Solutions and Dilutions
.50
2.7
Buffers and
pH.52
VII
2.8
Appendix
Calculating
Titration
Curves for Polyprotic Acids and Other
Multiple Binding Site Receptors
.62
2.9
Equipment Used in This Course
.64
CHAPTER
3:
SPECTROSCOPIC METHODS
65
3.1
Introduction
.65
3.2
Design and Properties of Spectrophotometers
.69
3.3
Effects of Spectral Bandpass and Stray Light
.72
3.4
Recording Spectrophotometers
.74
3.5
Fluorescence Spectroscopy
.75
3.6
Chromogenic and Fluorogenic Reactions Used for Analysis
.83
3.7
Other
Spectroscopie
Techniques
.84
3.8
Mass Spectrometry (MS)
.94
Experiments
3-1
to
3-4.99
Reagents Needed for Chapter
3.103
CHAPTER
4:
QUANTIFICATION OF PROTEIN CONCENTRATION
105
4.1
Purposes of Protein Quantification
.105
4.2
Factors to Consider in Choosing an Assay
.107
4.3
Non-Colorimetric Procedures for Quantification of Proteins
.108
4.4
Colorimetrie
Procedures for Quantification of Proteins
.110
Experiment
4-1.115
Reagents Needed for Chapter
4.119
VIII
CHAPTER
5:
CHROMATOGRAPHY
121
5.1
Introduction
.121
5.2
Gel-Filtration (Size Exclusion or Gel-Permeation) Chromatography
.125
5.3
Affinity Chromatography
.133
5.4
Ion-Exchange Chromatography
.143
5.5 Hydrophobie
Interaction Chromatography
.149
Experiments
5-1
and
5-2.152
Reagents Needed for Chapter
5.160
CHAPTER
6:
GEL ELECTROPHORESIS OF PROTEINS
161
6.1
Process of Electrophoresis
.161
6.2
Polyacrylamide Gels
.163
6.3
SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) of Proteins
.165
6.4
Detection of Proteins in SDS-Polyacrylamide Gels
.171
6.5
Applications of SDS-PAGE
.173
Experiments
6-1
and
6-2.183
Reagents Needed for Chapter
6.190
CHAPTER
7:
OVERVIEW OF PROTEIN PURIFICATION
191
7.1
Introduction
.191
7.2
Development of a Suitable Assay Procedure
.193
7.3
Time, Temperature, and Yield
.195
7.4
Selection of the Best Source Material
.195
7.5
Solubilization of the Protein
.198
ix
7.6 Initial Steps
of Purification
.199
7.7
Developing a Series of High-Resolution
Chromatographie
Steps
.201
7.8
Methods Used to Change Buffer and Concentrate Protein Samples
.204
7.9
A Logical Series of Steps
.205
7.10
Storage of the Purified Protein
.205
7.11
The Protein Purification Table
.206
CHAPTER
8:
SUBCELLULAR FRACTIONATION
209
8.1
Introduction
.209
8.2
Structural Organization of Prokaryotic and Eukaryotic Cells
.210
8.3
Overview of Fractionation Protocols
.213
Experiment
8-1.220
Reagents Needed for Chapter
8.227
CHAPTER
9:
ISOLATION AND CHARACTERIZATION OF AN
ENZYME-ALKALINE PHOSPHATASE FROM ESCHERICHIA
COU
229
9.1
Objectives
.229
9.2
Introduction and Basic Principles
.229
9.3
Purification of Alkaline Phosphatase
.233
9.4
Characterization of Purified Alkaline Phosphatase
.242
Appendix
9-1
Assay of Alkaline Phosphatase
.243
Reagents and Equipment Needed for Chapter
9.247
CHAPTER
10: ENZYME
KINETICS
251
10.1
Why Use Steady-State Kinetics?
.251
10.2
Steady-State Kinetics Principles
.252
10.3
The Significance of Km and Vmax
.255
10.4
Graphical Analysis
.257
10.5
Competitive, "Noncompetitive,
"
and "Uncompetitive" Inhibitors
.260
Experiments
10-1
to
10-3.267
Reagents Needed for Chapter
10.273
CHAPTER
11 :
LIGAND BINDING
275
11.1
Ligand Binding Is the Key to Most Biological Processes
.275
11.2
Analysis of Ligand Binding at Equilibrium
.277
11.3
Digression on Regression
.283
11.4
Effects of the Concentrations of
L
and
R
.283
11.5
Effects of Two Sites and Cooperative Behavior
.285
11.6
Analysis of the Kinetics of Ligand Binding
.292
11.7
Methods Used to Study Receptor-Ligand Interactions
.295
Experiments
11-1
and
11-2.302
Reagents Needed for Chapter
11.308
CHAPTER
12:
ENZYMATIC METHODS OF ANALYSIS
309
12.1
Enzymatic Analysis of Substrates
.309
12.2
Assays for Enzymatic Activity
.310
12.3
Practical Considerations
.311
xi
12.4
Coupled Assays
.312
12.5
Experiments with
Pyridine
Nucleotide-Requiring Enzymes
.314
Experiments
12-1
to
12-4.317
Reagents Needed for Chapter
12.328
Appendix
12-1:
Sample Calculations
.330
CHAPTER
13:
RECOMBINANT
DNA
TECHNIQUES
337
13.1
Introduction
.337
13.2
Properties of Nucleic Acids
.339
13.3
Strategy of
Recombinant
DNA
Techniques
.341
13.4
Cutting and Splicing
DNA.343
13.5
Gel Electrophoresis of
DNA.347
13.6
Introducing
DNA
into Cells
.353
13.7
Identifying Transformed Cells
.354
13.8
Vectors, Hosts, and Libraries for
Recombinant
DNA
Experiments
.359
13.9
Applications of
Recombinant
DNA
Technology
.366
Experiments
13-1
to
13-4.375
Reagents Needed for Chapter
13.386
CHAPTER
14:
POLYMERASE CHAIN REACTION (PCR) TECHNOLOGY
389
14.1
Introduction
.389
14.2
Principle of the PCR Method
.390
14.3
The Three Steps of PCR Are Controlled by Temperature
.392
14.4
Applications of PCR
.394
xii
14.5 Legal
and Ethical Issues
.410
Experiments
14-1
to
14-6.412
Reagents Needed for Chapter
14.423
CHAPTER
15:
USING THE COMPUTER AND THE INTERNET
FOR BIOCHEMICAL RESEARCH AND COMMUNICATION
425
15.1
Introduction to the World Wide Web (the Internet)
.425
15.2
Information on the Internet Useful to Biochemists
.431
15.3
Literature and Data Searches
.432
15.4
Visualization of Molecular Structures
.440
15.5
Other Useful Information
.442
15.6
Homework Assignment
.445
INDEX
447
XIII |
| adam_txt |
Contents
PREFACE
xv
CHAPTER
1 :
GETTING STARTED IN SCIENTIFIC RESEARCH
1
1.1
The Difference Between Experiments and Demonstrations
.1
1.2
Philosophy and Design of Experiments
.3
1.3
Designing Informative Experiments
.4
1.4
Ethics in Science
.5
1.5
Keeping a Laboratory Notebook
.9
1.6
Laboratory Reports
.13
1.7
Presentation and Analysis of Data
.15
1.8
The
Minisymposium
.18
CHAPTER
2:
BASIC PROCEDURES IN THE
BIOCHEMISTRY LABORATORY
21
2.1
Laboratory Safety
.21
2.2
Special Safety Procedures Are Required for Using Radioactive
Materials or Operating the Autoclave
.26
2.3
Measurement of Weights, Volumes, and
pH.30
2.4
Various Instruments Used
.38
2.5
Other General Techniques
.45
2.6
Solutions and Dilutions
.50
2.7
Buffers and
pH.52
VII
2.8
Appendix
Calculating
Titration
Curves for Polyprotic Acids and Other
Multiple Binding Site Receptors
.62
2.9
Equipment Used in This Course
.64
CHAPTER
3:
SPECTROSCOPIC METHODS
65
3.1
Introduction
.65
3.2
Design and Properties of Spectrophotometers
.69
3.3
Effects of Spectral Bandpass and Stray Light
.72
3.4
Recording Spectrophotometers
.74
3.5
Fluorescence Spectroscopy
.75
3.6
Chromogenic and Fluorogenic Reactions Used for Analysis
.83
3.7
Other
Spectroscopie
Techniques
.84
3.8
Mass Spectrometry (MS)
.94
Experiments
3-1
to
3-4.99
Reagents Needed for Chapter
3.103
CHAPTER
4:
QUANTIFICATION OF PROTEIN CONCENTRATION
105
4.1
Purposes of Protein Quantification
.105
4.2
Factors to Consider in Choosing an Assay
.107
4.3
Non-Colorimetric Procedures for Quantification of Proteins
.108
4.4
Colorimetrie
Procedures for Quantification of Proteins
.110
Experiment
4-1.115
Reagents Needed for Chapter
4.119
VIII
CHAPTER
5:
CHROMATOGRAPHY
121
5.1
Introduction
.121
5.2
Gel-Filtration (Size Exclusion or Gel-Permeation) Chromatography
.125
5.3
Affinity Chromatography
.133
5.4
Ion-Exchange Chromatography
.143
5.5 Hydrophobie
Interaction Chromatography
.149
Experiments
5-1
and
5-2.152
Reagents Needed for Chapter
5.160
CHAPTER
6:
GEL ELECTROPHORESIS OF PROTEINS
161
6.1
Process of Electrophoresis
.161
6.2
Polyacrylamide Gels
.163
6.3
SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) of Proteins
.165
6.4
Detection of Proteins in SDS-Polyacrylamide Gels
.171
6.5
Applications of SDS-PAGE
.173
Experiments
6-1
and
6-2.183
Reagents Needed for Chapter
6.190
CHAPTER
7:
OVERVIEW OF PROTEIN PURIFICATION
191
7.1
Introduction
.191
7.2
Development of a Suitable Assay Procedure
.193
7.3
Time, Temperature, and Yield
.195
7.4
Selection of the Best Source Material
.195
7.5
Solubilization of the Protein
.198
ix
7.6 Initial Steps
of Purification
.199
7.7
Developing a Series of High-Resolution
Chromatographie
Steps
.201
7.8
Methods Used to Change Buffer and Concentrate Protein Samples
.204
7.9
A Logical Series of Steps
.205
7.10
Storage of the Purified Protein
.205
7.11
The Protein Purification Table
.206
CHAPTER
8:
SUBCELLULAR FRACTIONATION
209
8.1
Introduction
.209
8.2
Structural Organization of Prokaryotic and Eukaryotic Cells
.210
8.3
Overview of Fractionation Protocols
.213
Experiment
8-1.220
Reagents Needed for Chapter
8.227
CHAPTER
9:
ISOLATION AND CHARACTERIZATION OF AN
ENZYME-ALKALINE PHOSPHATASE FROM ESCHERICHIA
COU
229
9.1
Objectives
.229
9.2
Introduction and Basic Principles
.229
9.3
Purification of Alkaline Phosphatase
.233
9.4
Characterization of Purified Alkaline Phosphatase
.242
Appendix
9-1
Assay of Alkaline Phosphatase
.243
Reagents and Equipment Needed for Chapter
9.247
CHAPTER
10: ENZYME
KINETICS
251
10.1
Why Use Steady-State Kinetics?
.251
10.2
Steady-State Kinetics Principles
.252
10.3
The Significance of Km and Vmax
.255
10.4
Graphical Analysis
.257
10.5
Competitive, "Noncompetitive,
"
and "Uncompetitive" Inhibitors
.260
Experiments
10-1
to
10-3.267
Reagents Needed for Chapter
10.273
CHAPTER
11 :
LIGAND BINDING
275
11.1
Ligand Binding Is the Key to Most Biological Processes
.275
11.2
Analysis of Ligand Binding at Equilibrium
.277
11.3
Digression on Regression
.283
11.4
Effects of the Concentrations of
L
and
R
.283
11.5
Effects of Two Sites and Cooperative Behavior
.285
11.6
Analysis of the Kinetics of Ligand Binding
.292
11.7
Methods Used to Study Receptor-Ligand Interactions
.295
Experiments
11-1
and
11-2.302
Reagents Needed for Chapter
11.308
CHAPTER
12:
ENZYMATIC METHODS OF ANALYSIS
309
12.1
Enzymatic Analysis of Substrates
.309
12.2
Assays for Enzymatic Activity
.310
12.3
Practical Considerations
.311
xi
12.4
Coupled Assays
.312
12.5
Experiments with
Pyridine
Nucleotide-Requiring Enzymes
.314
Experiments
12-1
to
12-4.317
Reagents Needed for Chapter
12.328
Appendix
12-1:
Sample Calculations
.330
CHAPTER
13:
RECOMBINANT
DNA
TECHNIQUES
337
13.1
Introduction
.337
13.2
Properties of Nucleic Acids
.339
13.3
Strategy of
Recombinant
DNA
Techniques
.341
13.4
Cutting and Splicing
DNA.343
13.5
Gel Electrophoresis of
DNA.347
13.6
Introducing
DNA
into Cells
.353
13.7
Identifying Transformed Cells
.354
13.8
Vectors, Hosts, and Libraries for
Recombinant
DNA
Experiments
.359
13.9
Applications of
Recombinant
DNA
Technology
.366
Experiments
13-1
to
13-4.375
Reagents Needed for Chapter
13.386
CHAPTER
14:
POLYMERASE CHAIN REACTION (PCR) TECHNOLOGY
389
14.1
Introduction
.389
14.2
Principle of the PCR Method
.390
14.3
The Three Steps of PCR Are Controlled by Temperature
.392
14.4
Applications of PCR
.394
xii
14.5 Legal
and Ethical Issues
.410
Experiments
14-1
to
14-6.412
Reagents Needed for Chapter
14.423
CHAPTER
15:
USING THE COMPUTER AND THE INTERNET
FOR BIOCHEMICAL RESEARCH AND COMMUNICATION
425
15.1
Introduction to the World Wide Web (the Internet)
.425
15.2
Information on the Internet Useful to Biochemists
.431
15.3
Literature and Data Searches
.432
15.4
Visualization of Molecular Structures
.440
15.5
Other Useful Information
.442
15.6
Homework Assignment
.445
INDEX
447
XIII |
| any_adam_object | 1 |
| any_adam_object_boolean | 1 |
| author | Ninfa, Alexander J. Ballou, David P. Benore, Marilee |
| author_facet | Ninfa, Alexander J. Ballou, David P. Benore, Marilee |
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| author_sort | Ninfa, Alexander J. |
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| ctrlnum | (OCoLC)420027217 (DE-599)DNB98756482X |
| dewey-full | 572.078 |
| dewey-hundreds | 500 - Natural sciences and mathematics |
| dewey-ones | 572 - Biochemistry |
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| dewey-search | 572.078 |
| dewey-sort | 3572.078 |
| dewey-tens | 570 - Biology |
| discipline | Biologie |
| discipline_str_mv | Biologie |
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| id | DE-604.BV023169603 |
| illustrated | Illustrated |
| index_date | 2024-07-02T19:57:03Z |
| indexdate | 2024-07-20T09:32:51Z |
| institution | BVB |
| isbn | 9780470087664 |
| language | English |
| oai_aleph_id | oai:aleph.bib-bvb.de:BVB01-016356285 |
| oclc_num | 420027217 |
| open_access_boolean | |
| owner | DE-703 DE-29T DE-19 DE-BY-UBM DE-11 DE-188 |
| owner_facet | DE-703 DE-29T DE-19 DE-BY-UBM DE-11 DE-188 |
| physical | XIX, 458 S. Ill., graph. Darst. |
| publishDate | 2010 |
| publishDateSearch | 2010 |
| publishDateSort | 2010 |
| publisher | Wiley |
| record_format | marc |
| spelling | Ninfa, Alexander J. Verfasser aut Fundamental laboratory approaches for biochemistry and biotechnology Alexander J. Ninfa ; David P. Ballou ; Marilee Benore 2. ed. Hoboken, NJ Wiley 2010 XIX, 458 S. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Biochemistry Laboratory Manuals Biochemistry Laboratory manuals Biotechnology Laboratory Manuals Biotechnology Laboratory manuals Molecular Biology Laboratory Manuals Methode (DE-588)4038971-6 gnd rswk-swf Biotechnologie (DE-588)4069491-4 gnd rswk-swf Biologisches Verfahren (DE-588)4361950-2 gnd rswk-swf Biochemische Methode (DE-588)4319880-6 gnd rswk-swf Biochemie (DE-588)4006777-4 gnd rswk-swf Bioverfahrenstechnik (DE-588)4307166-1 gnd rswk-swf Biochemische Methode (DE-588)4319880-6 s Biologisches Verfahren (DE-588)4361950-2 s Bioverfahrenstechnik (DE-588)4307166-1 s Biotechnologie (DE-588)4069491-4 s DE-604 Methode (DE-588)4038971-6 s 1\p DE-604 Biochemie (DE-588)4006777-4 s 2\p DE-604 Ballou, David P. Verfasser aut Benore, Marilee Verfasser aut text/html http://deposit.dnb.de/cgi-bin/dokserv?id=3070400&prov=M&dok_var=1&dok_ext=htm Inhaltstext Digitalisierung UB Bayreuth application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=016356285&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis 1\p cgwrk 20201028 DE-101 https://d-nb.info/provenance/plan#cgwrk 2\p cgwrk 20201028 DE-101 https://d-nb.info/provenance/plan#cgwrk |
| spellingShingle | Ninfa, Alexander J. Ballou, David P. Benore, Marilee Fundamental laboratory approaches for biochemistry and biotechnology Biochemistry Laboratory Manuals Biochemistry Laboratory manuals Biotechnology Laboratory Manuals Biotechnology Laboratory manuals Molecular Biology Laboratory Manuals Methode (DE-588)4038971-6 gnd Biotechnologie (DE-588)4069491-4 gnd Biologisches Verfahren (DE-588)4361950-2 gnd Biochemische Methode (DE-588)4319880-6 gnd Biochemie (DE-588)4006777-4 gnd Bioverfahrenstechnik (DE-588)4307166-1 gnd |
| subject_GND | (DE-588)4038971-6 (DE-588)4069491-4 (DE-588)4361950-2 (DE-588)4319880-6 (DE-588)4006777-4 (DE-588)4307166-1 |
| title | Fundamental laboratory approaches for biochemistry and biotechnology |
| title_auth | Fundamental laboratory approaches for biochemistry and biotechnology |
| title_exact_search | Fundamental laboratory approaches for biochemistry and biotechnology |
| title_exact_search_txtP | Fundamental laboratory approaches for biochemistry and biotechnology |
| title_full | Fundamental laboratory approaches for biochemistry and biotechnology Alexander J. Ninfa ; David P. Ballou ; Marilee Benore |
| title_fullStr | Fundamental laboratory approaches for biochemistry and biotechnology Alexander J. Ninfa ; David P. Ballou ; Marilee Benore |
| title_full_unstemmed | Fundamental laboratory approaches for biochemistry and biotechnology Alexander J. Ninfa ; David P. Ballou ; Marilee Benore |
| title_short | Fundamental laboratory approaches for biochemistry and biotechnology |
| title_sort | fundamental laboratory approaches for biochemistry and biotechnology |
| topic | Biochemistry Laboratory Manuals Biochemistry Laboratory manuals Biotechnology Laboratory Manuals Biotechnology Laboratory manuals Molecular Biology Laboratory Manuals Methode (DE-588)4038971-6 gnd Biotechnologie (DE-588)4069491-4 gnd Biologisches Verfahren (DE-588)4361950-2 gnd Biochemische Methode (DE-588)4319880-6 gnd Biochemie (DE-588)4006777-4 gnd Bioverfahrenstechnik (DE-588)4307166-1 gnd |
| topic_facet | Biochemistry Laboratory Manuals Biochemistry Laboratory manuals Biotechnology Laboratory Manuals Biotechnology Laboratory manuals Molecular Biology Laboratory Manuals Methode Biotechnologie Biologisches Verfahren Biochemische Methode Biochemie Bioverfahrenstechnik |
| url | http://deposit.dnb.de/cgi-bin/dokserv?id=3070400&prov=M&dok_var=1&dok_ext=htm http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=016356285&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
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