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University of New Orleans Theses and
Dissertations and Theses
Dissertations
Spring 5-13-2016
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Janet Gonzalez-Rosario
University of New Orleans, New Orleans, [email protected]
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Gonzalez-Rosario, Janet, "Purification and characterization of antibodies against killifish HIF-1α" (2016).
University of New Orleans Theses and Dissertations. 2150.
https://scholarworks.uno.edu/td/2150
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Purification and characterization of antibodies against killifish HIF-1
A Thesis
Submitted to the Graduate Faculty of the
University of New Orleans
in partial fulfillment of the
requirements for the degree of
Master of Science
in
Biological Sciences
by
Janet Gonzalez-Rosario
B.S. University of New Orleans, 2013
May 2016
Copyright 2016
Janet Gonzalez-Rosario
ii
DEDICATION
This thesis is dedicated to my parents, my husband and kids, who have supported me along the
journey.
iii
ACKNOWLEDGEMENTS
Graduate school was an incredible journey of personal growth and professional development.
Thanks to the department of Biological Sciences for this opportunity. My advisor, Dr. Bernard B.
Rees, is the reason this thesis is possible. He took a chance on me when he invited me into his
lab as an undergraduate student and then believed in me enough to let me stay to do a Masters.
He has been an incredible teacher, mentor, advisor, and friend during my time at the University
of New Orleans. I would also like to express my appreciation to Dr. Wendy Schluchter. She was
the first person I met when I came to UNO as a transfer student, and has been an invaluable
advisor, mentor, and teacher. Dr. Schluchter, along with Dr. Carla Penz, selected me for the
Undergraduate, Research and Mentoring Award that introduced me to research and led the way
to this thesis. Dr. Schluchter and Dr. Zhengchang Liu for helping to guide me through this
research project and agreeing without hesitation to be part of my thesis committee. I am grateful
to all that contributed in any way towards helping me with various aspects of my thesis. This
includes all my lab mates and fellow graduate students who were always supportive. While
working on her own thesis, Miss Jenna D. Hill allowed me to be her shadow when I started as an
undergraduate research assistant. Miss Kristina M. Farragut, Mr. Luis Matute, Mr. Joe Diaz, and
Miss Christina M. Kronfel, have been great friends and helped with various experiments. Mr.
Dillon Chung working in Dr. Patricia Schulte’s laboratory at the University of British Columbia
for growing and preparing all the cell cultures used in experiments. Thanks to Dr. Yang Cai from
the Children’s Hospital and the Proteomics Core at the University of British Columbia, Canada
for running my mass spectrometry samples. I would like to say thank you to my family, who are
the most proud of me, and are equally worthy of acknowledgement. My parents Jose and Olga
Gonzalez who taught me that with hard work and perseverance, anything is possible. My
iv
amazing husband Michael S. Rosario, who is my best friend and partner. His support and
encouragement allowed me to follow my dreams. My children, Samantha, Vanessa, and Michael
L. Rosario for reminding me why this journey was important.
v
CONTENTS
LIST OF FIGURES ..................................................................................................................... viii
LIST OF TABLES ......................................................................................................................... ix
ABSTRACT .....................................................................................................................................x
INTRODUCTION ...........................................................................................................................1
1.1 Oxygen ...........................................................................................................................1
1.2 Hypoxia Inducible Factors .............................................................................................2
1.3 The Aquatic Environment ..............................................................................................3
1.4 Fish and HIF ..................................................................................................................5
1.5 Antibodies ......................................................................................................................6
1.6 Fundulus as a Model System .........................................................................................7
1.7 Research Goals...............................................................................................................7
MATERIALS AND METHODS .....................................................................................................9
2.1 Anti-HIF-1 IgY ............................................................................................................9
2.2 Cloning, Expression, and Purification of Polyhistidine-tagged HIF-1 Peptide ..........9
2.3 Protein Assay, Electrophoresis, and Western Blotting ................................................13
2.4 Destaining and Digestion of Gel Slices for Mass Spectrometry..................................15
2.5 Affinity Purification of HIF-1 Antibodies .................................................................17
2.6 Immunoprecipitation ....................................................................................................18
2.7 Cell Culture Experiments .............................................................................................19
2.8 Animal Exposure and Tissue Lysates ..........................................................................21
RESULTS ......................................................................................................................................23
3.1 Cloning, Expression, and Purification of Polyhistidine-tagged HIF-1 Peptide ........23
vi
3.2 Antibody Purification of HIF-1 Antibodies ..............................................................24
3.3 Immunoprecipitation Optimization ..............................................................................25
3.4 Immunoprecipitation of HIF-1 from KFE-5 cell extracts .........................................26
3.5 Immunoprecipitation of HIF-1 from F. grandis tissues ............................................29
DISCUSSION ................................................................................................................................31
REFRENCES .................................................................................................................................35
VITA ............................................................................................................................................58
vii
LIST OF FIGURES
Figure 1: Schematic representation of HIF- domains .................................................................42
Figure 2: Oxygen-dependence of HIF abundance and function ....................................................43
Figure 3: HIF-1 c-DNA sequence ...............................................................................................44
Figure 4: Results from Ni-NTA and ion-exchange chromatography ............................................45
Figure 5: Results from Ni-NTA, ion-exchange, and size-exclusion chromatography ..................46
Figure 6: Overall HIF- recombinant protein purification ...........................................................47
Figure 7: Dot-blot of antibody purification ...................................................................................48
Figure 8: Western blot of IVTT HIF- proteins ............................................................................49
Figure 9: Optimization of immunoprecipitation variables.............................................................50
Figure 10: Immunoprecipitation of KFE-5 cell extracts ................................................................51
Figure 11: Immunoprecipitation of KFE-5 cell extracts after CoCl stimulation ..........................52
2
Figure 12: Immunoprecipitation of KFE-5 cytoplasmic and nuclear extracts ...............................53
Figure 13: Total protein gel of KFE-5 cytoplasmic and nuclear extracts ......................................54
Figure 14: Immunoprecipitation of normoxic F. grandis tissue extracts ......................................55
Figure 15: Immunoprecipitation of hypoxic F. grandis tissue extracts .........................................56
Figure 16: Immunoprecipitation of normoxic and hypoxic F. grandis tissue extracts ..................57
viii
LIST OF TABLES
Table 1: Mass spectrometry results of protein purification fractions ............................................40
Table 2: Purification of HIF-1 from 4 x 1-liter E. coli cultures ..................................................41
ix
Description:This thesis is dedicated to my parents, my husband and kids, who have .. Figure 14: Immunoprecipitation of normoxic F. grandis tissue extracts .
