Table Of ContentBiochemistry in the Lab
A Manual for Undergraduates
Biochemistry in the Lab
A Manual for Undergraduates
by
Benjamin F. Lasseter
CRC Press
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Library of Congress Cataloging-in-Publication Data
Names: Lasseter, Benjamin F., author.
Title: Biochemistry in the lab : a manual for undergraduates / by Benjamin
F. Lasseter.
Description: Boca Raton, Florida : CRC Press, [2020] | Includes
bibliographical references and index. | Summary: “Biochemistry in the
Lab: A Manual for Undergraduates presents a comprehensive approach to
modern biochemistry laboratory teaching together with a complete
experimental experience, from molecular biology, cloning, and protein
expression, to purification and characterization”-- Provided by publisher.
Identifiers: LCCN 2019026897 (print) | LCCN 2019026898 (ebook) | ISBN
9781138589964 (paperback) | ISBN 9781138589926 (hardback) | ISBN
9780429491269 (ebook)
Subjects: LCSH: Biochemistry--Laboratory manuals.
Classification: LCC QD415.5 .L37 2020 (print) | LCC QD415.5 (ebook) | DDC
572.078--dc23
LC record available at https://lccn.loc.gov/2019026897
LC ebook record available at https://lccn.loc.gov/2019026898
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Contents Contents
This book was written for Sumie and the lads.
Here’s tae us. Wha’s like us? Ōku wa watashitachi no yōde
wa arimasen, soshite sorera no subete ga shinde imasu!
Contents
Author Bio ..............................................................................................................xiii
Chapter 1 Buffers ..................................................................................................1
Making Buffers ....................................................................................2
Method 1: A Free Acid Titrated by NaOH ......................................2
Method 2: A Free Base Titrated by HCl..........................................2
Method 3: Adding Free Acid and Free Base to Make the Buffer. .......3
Concentrated Buffers............................................................................4
Goal ......................................................................................................5
Optional Goal .......................................................................................6
Pre-Lab Questions ................................................................................6
Post-Lab Questions ...............................................................................7
Chapter 2 Assays ...................................................................................................9
1. Lactate Dehydrogenase: Assay by Substrates Alone .......................9
2. Phosphofructokinase: Assay by Metabolic Pathway .....................10
3. Assay by Immunochemistry ..........................................................12
4. Catalase: Assay by Physical Properties .........................................12
Ways of Reporting Enzyme Activity .............................................13
Evaluating a Source for an Enzyme ..............................................14
Serial Dilutions ..............................................................................14
Goal ...............................................................................................15
Materials ........................................................................................15
Instructions ....................................................................................16
Pre-Lab Questions ..............................................................................19
Post-Lab Questions .............................................................................20
Chapter 3 Protein Concentration.........................................................................21
1. Absorbance at 280 nm ...................................................................21
2. The Bradford Assay.......................................................................22
3. Bicinchionic Acid Assay ...............................................................23
Finding the Concentration ..................................................................23
Goal ...............................................................................................26
Materials ........................................................................................26
Instructions ....................................................................................26
Recipe for Bradford Reagent .........................................................28
Pre-Lab Questions ..............................................................................28
Post-Lab Questions .............................................................................29
vii
viii Contents
Chapter 4 ELISA .................................................................................................31
Goal ....................................................................................................33
Materials .............................................................................................33
Instructions .........................................................................................33
Pre-Lab Questions ..............................................................................34
Post-Lab Questions .............................................................................35
Chapter 5 Salting Out Proteins and Other Biomolecules ...................................37
Goal ....................................................................................................40
Method ...........................................................................................40
Data Analysis .....................................................................................41
Pre-Lab Questions ..............................................................................42
Post-Lab Questions .............................................................................42
Chapter 6 A Discussion of Isoelectric Point and Effective Charge ....................45
Effective Charge .................................................................................45
Isoelectric Points ................................................................................47
Questions ............................................................................................50
Chapter 7 Column Chromatography ...................................................................53
Types of Chromatography ..................................................................54
Backbone Issues .................................................................................59
Goal ....................................................................................................60
Pre-Lab Questions ..............................................................................65
Post-Lab Questions .............................................................................66
Chapter 8 Michaelis–Menten Kinetics ...............................................................69
Cooperativity ......................................................................................73
Data in Triplicate ................................................................................75
Goal ...............................................................................................75
Procedure .......................................................................................76
Catalase: A Simple Assay .........................................................76
Pre-Lab Questions ..............................................................................78
Post-Lab Questions .............................................................................79
Chapter 9 Protein Purification ............................................................................81
Dialysis ...............................................................................................81
Purification Tables ..............................................................................82
Goal ....................................................................................................85
Method ...............................................................................................85
First Lab Session ...........................................................................85
Second Lab Session .......................................................................86
Third Lab Session ..........................................................................86
Contents ix
Pre-Lab Questions ..............................................................................86
Post-Lab Questions .............................................................................87
Chapter 10 Polyacrylamide Gels ...........................................................................89
Acrylamide Chemistry Affects Separations ......................................89
Buffering Gels ....................................................................................91
Goal ...............................................................................................92
Method ...........................................................................................92
Loading and Running an SDS-PAGE Gel..........................................93
Goal (Week 2) ................................................................................93
Method ...........................................................................................94
Pre-Lab Questions ..............................................................................96
Post Lab Questions .............................................................................98
Chapter 11 In Silico Biochemistry: The Evolution of Globins ..........................101
Globins .............................................................................................101
BLOSUM .........................................................................................102
Aligning and Scoring Sequences .....................................................106
Gap Penalties ....................................................................................106
Alignment Tools ...............................................................................107
Calculating Evolutionary Distance...................................................108
Goal .............................................................................................111
Post-Lab Questions ...........................................................................114
References ........................................................................................114
Chapter 12 Growing Crystals of Hemoglobin ....................................................115
Hanging Drop and Sitting Drop Methods ........................................116
Refining Crystal Growth ..................................................................119
Other Issues ......................................................................................120
Objective ...........................................................................................121
Procedure .....................................................................................121
Materials .................................................................................121
Pre-Lab Questions ............................................................................126
Post-Lab Questions ...........................................................................126
Chapter 13 Enzyme Inhibition ............................................................................127
Competitive Inhibition .....................................................................127
Uncompetitive Inhibition ..................................................................129
Mixed Inhibition...............................................................................131
Acid Phosphatase .............................................................................132
Objective ...........................................................................................133
Materials ......................................................................................134
Post-Lab Questions ...........................................................................136
Reference ..........................................................................................136
