Experiments in
Biochemistry:
A Hands On Approach
i
, Instructor's Manual for
Experiments in
Biochemistry:
A Hands On Approach
Shawn O. Farrell
and
Lynn E. Taylor
Colorado State University
ii
Experiments in Biochemistry: A Hands-on Approach, 2nd Edition Shawn O. Farrell
Colorado, Lynn E. Taylor
, Table of Contents
Preface........................................................................................................................iii
Technical Support......................................................................................................iv
Background Information from Textbooks.................................................................v
General Lab Equipment.............................................................................................vi
Experiment 1 -- Use of pipettors................................................................................1
Experiment 2 -- Preparation of Buffers.....................................................................7
Experiment 3 -- Beer's Law and Standard Curves.....................................................14
Experiment 3a -- Protein Concentration of LDH Fractions.......................................20
Experiment 4 -- Purification of LDH (short version)................................................25
Experiment 4a -- Purification of LDH (comprehensive version)..............................33
Experiment 5 -- Separation and Identification of Amino Acids................................42
Experiment 5a -- Purification of LDH with Ion Exchange Chromatography............49
Experiment 6 -- Affinity Chromatography of LDH...................................................57
Experiment 6a – Affinity Chromatography of LDH..................................................61
Experiment 7 -- Gel Filtration Chromatography.......................................................69
Experiment 7a -- Gel Filtration Chromatography of LDH........................................74
Experiment 8 -- Enzyme Kinetics of Tyrosinase.......................................................82
Experiment 8a -- Enzyme Kinetics of LDH..............................................................89
Experiment 9 -- Native Gel Separation of LDH Isozymes (short version)................96
Experiment 9a -- Native Gel Separation of LDH Isozymes (comprehensive)..........101
Experiment 9b -- SDS-Polyacrylamide Gel Electrophoresis (short).........................106
Experiment 9c -- SDS-Polyacrylamide Gel Electrophoresis (comprehensive).........112
Experiment 10 -- Western Blots of Serum Proteins..................................................119
Experiment 10a -- Western Blot of LDH...................................................................127
Experiment 11 -- Analysis of DNA Restriction Fragments.......................................134
Experiment 12 -- Cloning and Expression of Barracuda LDH..................................141
Experiment 13 -- Polymerase Chain Reaction...........................................................158
iii
, Preface
This manual is designed to aid the laboratory instructor in preparing for, supervising, and
analyzing experiments in Experimental Biochemistry: A Hands On Approach. The text
was written with the idea of its application to a wide variety of biochemistry lab courses.
Whether your course is for non-majors and meets only once a week for 3 hours, or is a
more intensive course requiring several meetings, you should be able to find the
appropriate length and intensity of experiments. Many of the experiments are suitable for
a survey course and can stand alone. For those interested in a comprehensive approach,
there are groups of experiments that do make an integrated package. Many of the
techniques used offer both types of experiment. For example, chapter 7 involves gel
filtration chromatography. Experiment 7 is a stand alone experiment involving estimating
the molecular weight of myoglobin with Sephadex G-75. Experiment 7a is part of the
comprehensive purification of lactate dehydrogenase and uses gel filtration on Sephadex
G-150 or Sephacryl S-200 as the final purification step.
Each chapter of this manual corresponds to the same chapter in Experiments in
Biochemistry and includes the following:
Estimated time required by competent and prepared students
Materials needed for the experiment and sources when necessary
Hints for the preparation of reagents and equipment, and pitfalls to avoid
Suggestions for disposal and clean up of experiments
Sample data the students are likely to see and answers to the end of experiment
calculations
Answers to the additional problem sets.
We hope that armed with this manual you will find the implementation of the
experiments straight forward. If you ever have trouble, please feel free to contact the
author (see Tech Support).
iv
Biochemistry:
A Hands On Approach
i
, Instructor's Manual for
Experiments in
Biochemistry:
A Hands On Approach
Shawn O. Farrell
and
Lynn E. Taylor
Colorado State University
ii
Experiments in Biochemistry: A Hands-on Approach, 2nd Edition Shawn O. Farrell
Colorado, Lynn E. Taylor
, Table of Contents
Preface........................................................................................................................iii
Technical Support......................................................................................................iv
Background Information from Textbooks.................................................................v
General Lab Equipment.............................................................................................vi
Experiment 1 -- Use of pipettors................................................................................1
Experiment 2 -- Preparation of Buffers.....................................................................7
Experiment 3 -- Beer's Law and Standard Curves.....................................................14
Experiment 3a -- Protein Concentration of LDH Fractions.......................................20
Experiment 4 -- Purification of LDH (short version)................................................25
Experiment 4a -- Purification of LDH (comprehensive version)..............................33
Experiment 5 -- Separation and Identification of Amino Acids................................42
Experiment 5a -- Purification of LDH with Ion Exchange Chromatography............49
Experiment 6 -- Affinity Chromatography of LDH...................................................57
Experiment 6a – Affinity Chromatography of LDH..................................................61
Experiment 7 -- Gel Filtration Chromatography.......................................................69
Experiment 7a -- Gel Filtration Chromatography of LDH........................................74
Experiment 8 -- Enzyme Kinetics of Tyrosinase.......................................................82
Experiment 8a -- Enzyme Kinetics of LDH..............................................................89
Experiment 9 -- Native Gel Separation of LDH Isozymes (short version)................96
Experiment 9a -- Native Gel Separation of LDH Isozymes (comprehensive)..........101
Experiment 9b -- SDS-Polyacrylamide Gel Electrophoresis (short).........................106
Experiment 9c -- SDS-Polyacrylamide Gel Electrophoresis (comprehensive).........112
Experiment 10 -- Western Blots of Serum Proteins..................................................119
Experiment 10a -- Western Blot of LDH...................................................................127
Experiment 11 -- Analysis of DNA Restriction Fragments.......................................134
Experiment 12 -- Cloning and Expression of Barracuda LDH..................................141
Experiment 13 -- Polymerase Chain Reaction...........................................................158
iii
, Preface
This manual is designed to aid the laboratory instructor in preparing for, supervising, and
analyzing experiments in Experimental Biochemistry: A Hands On Approach. The text
was written with the idea of its application to a wide variety of biochemistry lab courses.
Whether your course is for non-majors and meets only once a week for 3 hours, or is a
more intensive course requiring several meetings, you should be able to find the
appropriate length and intensity of experiments. Many of the experiments are suitable for
a survey course and can stand alone. For those interested in a comprehensive approach,
there are groups of experiments that do make an integrated package. Many of the
techniques used offer both types of experiment. For example, chapter 7 involves gel
filtration chromatography. Experiment 7 is a stand alone experiment involving estimating
the molecular weight of myoglobin with Sephadex G-75. Experiment 7a is part of the
comprehensive purification of lactate dehydrogenase and uses gel filtration on Sephadex
G-150 or Sephacryl S-200 as the final purification step.
Each chapter of this manual corresponds to the same chapter in Experiments in
Biochemistry and includes the following:
Estimated time required by competent and prepared students
Materials needed for the experiment and sources when necessary
Hints for the preparation of reagents and equipment, and pitfalls to avoid
Suggestions for disposal and clean up of experiments
Sample data the students are likely to see and answers to the end of experiment
calculations
Answers to the additional problem sets.
We hope that armed with this manual you will find the implementation of the
experiments straight forward. If you ever have trouble, please feel free to contact the
author (see Tech Support).
iv
