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E-grāmata: Biochemistry in the Lab: A Manual for Undergraduates

(Christopher Newport University, VA, USA)
  • Formāts: 168 pages
  • Izdošanas datums: 30-Sep-2019
  • Izdevniecība: CRC Press
  • Valoda: eng
  • ISBN-13: 9780429957383
  • Formāts - PDF+DRM
  • Cena: 62,60 €*
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  • Formāts: 168 pages
  • Izdošanas datums: 30-Sep-2019
  • Izdevniecība: CRC Press
  • Valoda: eng
  • ISBN-13: 9780429957383

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Most lab manuals assume a high level of knowledge among biochemistry students, as well as a large amount of experience combining knowledge from separate scientific disciplines. Biochemistry in the Lab: A Manual for Undergraduates expects little more than basic chemistry. It explains procedures clearly, as well as giving a clear explanation of the theoretical reason for those steps.

Key Features:











Presents a comprehensive approach to modern biochemistry laboratory teaching, together with a complete experimental experience





Includes chemical biology as its foundation, teaching readers experimental methods specific to the field





Provides instructor experiments that are easy to prepare and execute, at comparatively low cost





Supersedes existing, older texts with information that is adjusted to modern experimental biochemistry





Is written by an expert in the field

This textbook presents a foundational approach to modern biochemistry laboratory teaching together with a complete experimental experience, from protein purification and characterization to advanced analytical techniques. It has modules to help instructors present the techniques used in a time critical manner, as well as several modules to study protein chemistry, including gel techniques, enzymology, crystal growth, unfolding studies, and fluorescence. It proceeds from the simplest and most important techniques to the most difficult and specialized ones. It offers instructors experiments that are easy to prepare and execute, at comparatively low cost.

Recenzijas

Author Lasseter (biochemistry, Christopher Newport Univ.) has written several laboratory manuals for general chemistry. This manual provides 18 chapters, each dealing with a single biochemical laboratory procedure. Several chapters end with a goalassemble the methods and materials to useand an optional goaluse and test materials after they are prepared. All chapters end with pre-lab and post-lab questions as well as references. Topics covered include buffers, assays, protein concentrations, ELISA (Enzyme-Linked ImmunoabSorption Assay), salting out biomolecules, isoelectric points and effective charges, column chromatography, Michaelis-Menten kinetics (biochemical reaction rates), protein purification, polyacrylamide gels and their uses, computational biochemistry (the example used is the evolution of globins), growing crystals of hemoglobin, enzyme inhibition, multisubstrate kinetics, fluorescence and denaturation, fluorescence studies of ligand binding, DNA restriction digests (i.e., determination of sites), and western blotting (combined use of ELISA and immunostaining). In the current pandemic time, applicability of any lab manual given total shutdown of academic labs followed by only partial reopening is problematic, yet resources are available for teaching science labs under these conditions. Undergraduate students of biochemistry and their instructors will appreciate this book given the appropriate conditions for use.

Summing Up: Recommended. Lower- and upper-division undergraduates. Students enrolled in two-year technical programs. Graduate students and faculty.

--R. E. Buntrock, independent scholar, CHOICE

Author Bio xiii
Chapter 1 Buffers
1(8)
Making Buffers
2(2)
Method 1 A Free Acid Titrated by NaOH
2(1)
Method 2 A Free Base Titrated by HCl
2(1)
Method 3 Adding Free Acid and Free Base to Make the Buffer
3(1)
Concentrated Buffers
4(1)
Goal
5(1)
Optional Goal
6(1)
Pre-Lab Questions
6(1)
Post-Lab Questions
7(2)
Chapter 2 Assays
9(12)
1 Lactate Dehydrogenase: Assay by Substrates Alone
9(1)
2 Phosphofructokinase: Assay by Metabolic Pathway
10(2)
3 Assay by Immunochemistry
12(1)
4 Catalase: Assay by Physical Properties
12(7)
Ways of Reporting Enzyme Activity
13(1)
Evaluating a Source for an Enzyme
14(1)
Serial Dilutions
14(1)
Goal
15(1)
Materials
15(1)
Instructions
16(3)
Pre-Lab Questions
19(1)
Post-Lab Questions
20(1)
Chapter 3 Protein Concentration
21(10)
1 Absorbance at 280 nm
21(1)
2 The Bradford Assay
22(1)
3 Bicinchionic Acid Assay
23(1)
Finding the Concentration
23(5)
Goal
26(1)
Materials
26(1)
Instructions
26(2)
Recipe for Bradford Reagent
28(1)
Pre-Lab Questions
28(1)
Post-Lab Questions
29(2)
Chapter 4 ELISA
31(6)
Goal
33(1)
Materials
33(1)
Instructions
33(1)
Pre-Lab Questions
34(1)
Post-Lab Questions
35(2)
Chapter 5 Salting Out Proteins and Other Biomolecules
37(8)
Goal
40(1)
Method
40(1)
Data Analysis
41(1)
Pre-Lab Questions
42(1)
Post-Lab Questions
42(3)
Chapter 6 A Discussion of Isoelectric Point and Effective Charge
45(8)
Effective Charge
45(2)
Isoelectric Points
47(3)
Questions
50(3)
Chapter 7 Column Chromatography
53(16)
Types of Chromatography
54(5)
Backbone Issues
59(1)
Goal
60(5)
Pre-Lab Questions
65(1)
Post-Lab Questions
66(3)
Chapter 8 Michaelis-Menten Kinetics
69(12)
Cooperativity
73(2)
Data in Triplicate
75(3)
Goal
75(1)
Procedure
76(1)
Catalase: A Simple Assay
76(2)
Pre-Lab Questions
78(1)
Post-Lab Questions
79(2)
Chapter 9 Protein Purification
81(8)
Dialysis
81(1)
Purification Tables
82(3)
Goal
85(1)
Method
85(1)
First Lab Session
85(1)
Second Lab Session
86(1)
Third Lab Session
86(1)
Pre-Lab Questions
86(1)
Post-Lab Questions
87(2)
Chapter 10 Polyacrylamide Gels
89(12)
Acrylamide Chemistry Affects Separations
89(2)
Buffering Gels
91(2)
Goal
92(1)
Method
92(1)
Loading and Running an SDS-PAGE Gel
93(3)
Goal (Week 2)
93(1)
Method
94(2)
Pre-Lab Questions
96(2)
Post Lab Questions
98(3)
Chapter 11 In Silico Biochemistry: The Evolution of Globins
101(14)
Globins
101(1)
BLOSUM
102(4)
Aligning and Scoring Sequences
106(1)
Gap Penalties
106(1)
Alignment Tools
107(1)
Calculating Evolutionary Distance
108(6)
Goal
111(3)
Post-Lab Questions
114(1)
References
114(1)
Chapter 12 Growing Crystals of Hemoglobin
115(12)
Hanging Drop and Sitting Drop Methods
116(3)
Refining Crystal Growth
119(1)
Other Issues
120(1)
Objective
121(5)
Procedure
121(1)
Materials
121(5)
Pre-Lab Questions
126(1)
Post-Lab Questions
126(1)
Chapter 13 Enzyme Inhibition
127(10)
Competitive Inhibition
127(2)
Uncompetitive Inhibition
129(2)
Mixed Inhibition
131(1)
Acid Phosphatase
132(1)
Objective
133(3)
Materials
134(2)
Post-Lab Questions
136(1)
Reference
136(1)
Chapter 14 Multisubstrate Kinetics
137(8)
Product Inhibition
137(2)
Experimental Design
139(1)
Objective
140(3)
Materials
140(3)
References
143(2)
Chapter 15 Fluorescence and Denaturation
145(14)
Denaturants
145(2)
Structural Studies by Fluorescence
147(3)
Fluorescence Resonance Energy Transfer (FRET)
150(1)
FRET Studies for Unfolding
151(1)
Experimental Design Considerations
151(1)
Fluorometer Design
151(1)
Inner Filter Effect
151(2)
Using an Unfolding Curve to Find Kfold and ΔGfold
153(3)
Goal
156(2)
Materials
156(1)
Method
156(2)
Post-Lab Questions
158(1)
Chapter 16 Fluorescence Studies of Ligand Binding
159(10)
Experimental Considerations
161(3)
Concentration Effects
161(1)
Ligand Fluorescence
161(1)
Fitting Data to a Loss of Intensity
161(3)
Goal
164(3)
Materials
164(1)
Method
165(2)
Data Analysis
167(1)
Post-Lab Questions
167(1)
References
168(1)
Chapter 17 DNA Restriction Digests
169(10)
Gene Expression
169(2)
Restriction Endonucleases
171(1)
Plasmid Types and Purposes
172(1)
Restriction Digests
173(3)
Goal
173(1)
Procedure
174(2)
Pre-Lab Questions
176(3)
Tutorial: How to Construct a Restriction Map, Logically
176(3)
Chapter 18 Western Blotting
179(6)
Goal
181(3)
Set-Up
181(1)
Materials
181(1)
Part 1
181(1)
Part 2
182(1)
Part 3
182(1)
Instructions
182(1)
Part 1 Native Gel Electrophoresis
182(1)
Part 2 Blotting
183(1)
Part 3 Immunostaining
183(1)
References
184(1)
Index 185
Dr. Benjamin F. Lasseter earned his Ph.D. in Biochemistry from Texas A&M University in 2003. He briefly assisted in the production of history textbooks for the Catholic Schools Textbook Project before teaching at a high school in Louisiana. He moved on to Christopher Newport University in 2011, where he teaches the biochemistry labs. He mixes analytical chemistry with pure biology to study environmental impacts of metal pollution.