DEVELOPMENT OF MICROSCALE SEPARATION AND BLOTTING METHODS FOR ANALYSIS OF SMALL-VOLUME BIOLOGICAL MIXTURES by Gwendolyn Judith Anderson A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Chemistry) in The University of Michigan 2012 Doctoral Committee: Professor Robert T. Kennedy, Chair Professor Mark E. Meyerhoff Professor Gary D. Smith Professor Shuichi Takayama © Gwendolyn Judith Anderson 2012 ! ! ACKNOWLEDGMENTS I would like to first acknowledge the support and guidance of my advisor, Dr. Bob Kennedy, as he immeasurably helped my progress as a scientist. Thank you for many thoughtful discussions and the occasional laugh! I am also grateful to my committee members, Drs. Meyerhoff, Smith and Takayama for the time necessary to serve on my committee. Your comments helped steer my research in new directions, which of course is always the point. Additionally, the day-to-day expertise and fellowship of my labmates has been a great source of strength, as I hope it will remain for a very many students in years to come. Drs. Chisolm and Clark helped orient me to instrumentation in the lab, for which I was always thankful. Drs. Perry, Payeur, and Evans (née Reid) did not play such a pivotal early role, but they sure made subsequent years a lot more fun. I also learned from working with Cynthia Cipolla, Erik Guetschow and Shi Jin on projects in the more recent past. All the rest were there too, and though it doesn’t do to keep count of favors, I am pretty sure I am in their debt. Friends and mentors along the way made the path much easier: Billy Clifford-Nunn, Dr. Michelle Bushey, Brendan Veeneman, Deborah Lepkowski, Ruthie C. Hasenfus, the cats, the dogs, and my classmates Thomas Slaney and Peng Song, many thanks. My gratitude spreads further to my immediate and extended family. I like to think I had something to do with the continuing legacy of science in my family, but really the credit should go to Dr. Baheru Yadeta, my uncle, who was very excited when I was able to tell him I was off to study at the University of Michigan. It seems quite long ago now, but he knew then that it was only the beginning, and was excited for me in that way he was famous for. Best of luck to my little sister Rachel! I admire her fortitude as she endures both summers in Austin and prolonged study of electrochemistry. Ben Franklin said, “Do not anticipate trouble, or worry about what may never happen. Keep in the sunlight.” When I think of Rachel, I think of sunshine. Maybe it’s the hair. ! ""! ! Lastly, I appreciate my parents, who have been staunchly supportive of this mysterious endeavor. Thank you always for seeking to understand me (!) and for copyediting large chunks of the following pages. You instilled in me an ancient lesson: what is worth doing is rarely easy, and so it is for each of us to carpe diem. ! ! """! ! TABLE OF CONTENTS ACKNOWLEDGMENTS .............................................................................................................. ii LIST OF FIGURES ....................................................................................................................... vi ABSTRACT................................................................................................................................... ix CHAPTER 1. Introduction ..............................................................................................................1 Western Blotting Background ......................................................................................................2 Principles of Capillary Electrophoresis and Capillary Gel Electrophoresis ................................6 Capillary Gel Electrophoresis of Proteins ..................................................................................10 Affinity Techniques in Capillary Electrophoresis......................................................................14 Commercial Products Related to Western Blotting....................................................................14 Specific Aims of Dissertation.....................................................................................................16 Chapter 2. Western Blotting Using Capillary Electrophoresis ......................................................20 Introduction ................................................................................................................................20 Experimental Methods................................................................................................................22 Results and Discussion...............................................................................................................26 Conclusion..................................................................................................................................35 Chapter 3. Improvements to CE-Western Blotting for Multiplexed Operation ...........................38 Introduction ................................................................................................................................38 Experimental Methods................................................................................................................47 Results and Discussion...............................................................................................................52 Conclusion..................................................................................................................................81 Chapter 4. Capillary Dot Blotting .................................................................................................84 Introduction ................................................................................................................................84 ! "#! ! Experimental Methods................................................................................................................88 Results and Discussion...............................................................................................................90 Conclusion................................................................................................................................105 Chapter 5. Conclusions and Future Directions ............................................................................109 Future Directions for CE-Westerns ..........................................................................................109 MULTI-SAMPLE CAPILLARY DOT BLOTTING.............................................................................111 ! "! ! LIST OF FIGURES Figure 1.1. Examples of recent CGE separations in capillaries.....................................................12 Figure 1.2. Glycerol effects on pore structures for sieving matrices in CGE................................13 Figure 1.3. Microfluidic chip schematic for high speed CGE .......................................................14 Figure 2.1. Instrument overview...................................................................................................27 Figure 2.2. Size-dependent separation of standard FITC-labeled proteins...................................27 Figure 2.3. Measurements of band broadening inside the sheath capillary and on membrane. ...30 Figure 2.4. CE-based Western blot of lysozyme at 3 different concentrations ............................31 Figure 2.5. Immunoassay of unlabeled sample proteins at low levels ..........................................32 Figure 2.6. Immunoassay of unlabeled peak with labeled size standards ....................................34 Figure 3.1: Instrument overview....................................................................................................50 Figure 3.2. Separation of fluorescent ladder in different background buffers...............................53 Figure 3.3. Commercial sieving media separations of protein standards in cell lysate after dialysis ...........................................................................................................................................54 Figure 3.4. Effect of t-ITP preconcentration on ladder separations conducted with online detection.........................................................................................................................................56 Figure 3.5. Preconcentration factor for tITP injections of different ladder concentrations...........57 Figure 3.6. Effect of polymer MW on CGE separations ...............................................................58 Figure 3.7. Ohm plots for commercial and two dextran gels as measured with the membrane capture instrument..........................................................................................................................59 Figure 3.8. Effect of ionic strength on ladder separations and dextran gel size-based separation performance ...................................................................................................................................60 Figure 3.9. Effect of dextran levels on CGE separations...............................................................62 ! "#! ! Figure 3.10. Effect of varied percentages of glycerol on ladder separations.................................63 Figure 3.11. Variations in buffers and additives to probe complexation mechanism ...................64 Figure 3.12. Summary of the best separation conditions for dextran gels.....................................64 Figure 3.13. Ohm plot testing and trial separation with new sieving formula ..............................67