The work in the dissertation expands the applications of DIEF and describes the development of incorporating DIEF in a microfluidic chip to create a comprehensive proteomics tool. Proof-of-concept DIEF experiments have been done previously, so the focus of this work is to explore the capabilities of DIEF. Dynamic isoelectric focusing (DIEF) is a separation technique invented by Dr. Luke Tolley. It is similar to capillary isoelectric focusing except it uses four high voltage electrodes to form a pH gradient instead of only two. The additional two electrodes are able to manipulate the pH gradient resulting in selection of the region and of the range of pH within a pre-defined sampling or extraction point. One of the first applications described for DIEF was to isolate a single protein from a complex mixture. The protein isolated was a cellulase enzyme capable of degrading multiple cellulose materials over a wide range of environmental conditions. DIEF did isolate the protein in a pH span of 0.005 which is equivalent to 0.075% of the total pH range. Fractions were collected for sequencing analysis, but the fractions were contaminated with keratin both times. DIEF was also successfully performed in an open air channel. Though other electromigration techniques have been successfully done in open air channels, these techniques were severely time and pH limited. In contrast, DIEF in an open air channel is capable of using the entire 3-10 pH range and can perform isolations until the proteins are completely separated. The device developed was also an improvement on increasing sample capacity. The channel was significantly bigger than the traditional glass capillaries used. Since the channel was open, fraction collection was made simpler by collecting using a pipette. This work also demonstrated that DIEF can be made through the use of silicone molding compounds and polyurethane. The amount of milling needed is reduced, the pieces are produced quickly, and a single mold can produce several pieces. Machining pieces with fragile bits is not needed to be done as much since only one acrylic piece is required produce a mold. The mold can produce several polyurethane pieces. This fabrication method has proven useful for making DIEF holders. The next step was to make DIEF a truly comprehensive proteomic tool by incorporating it into a microfluidic chip. Multiple sample fractions are rapidly generated on chip through the use of multiple bubbles simultaneously injected into the separation channel. This stops the separation and, since each droplet is isolated from others by a bubble on each side, the protein peaks are not able to broaden. This novel use of digital microfluidics is still a work in progress, but the fundamentals have been demonstrated. The fabrication protocol for making molds and PDMS casts was developed using materials and procedures that can be done in a common laboratory environment. DIEF is a separation technique still in its infancy, with a wide variety of available applications. DIEF will continue to be tested in other areas and developed into a comprehensive proteomic tool.

The aim of this Master Thesis is the optimization and verification of conditions of separation of proteins from biological materials. For the separation a developed technique called micropreparative isoelectric focusing is being used. The theoretical part describes analytical methods of biological materials with a focus on proteins which were used for the optimization of the developing system in general. This part of the Master Thesis also presents this method itself. Furthermore, basic characteristics of other techniques used to control the efficiency of the micropreparative isoelectric focusing are introduced. The experimental part depicts the individual steps of optimization of the micropreparative isoelectric focusing. The ideal separation procedure was firstly found out on the individual standard proteins, secondly on their mixture and lastly the optimised procedure was applied to a real biological sample. The efficiency of the micropreparative isoelectric focusing was verified by SDS-PAGE and MALDI-TOF mass spectrometry.

Allozyme frequencies and general protein patterns were surveyed among selected Texas marine fishes and shellfishes to illustrate the application of biochemical genetic techniques to stock and species identification in fisheries management.

Congenital disorders of glycosylation (CDG) represent a heterogeneous group of mul- tisystemic metabolic disorders which are caused by defects in biosynthetic pathways of glycoproteins. The screening test for N-glycosylation disorders is the analyses of sialylated isoforms of serum transferrin (Tf) by means of isoelectric focusing (IEF). Two distinct pathological IEF patterns of Tf are observed. A type I pattern is cha- racterized by a decrease of tetra- and an increase of di- and asialotransferrin, whereas a type II pattern shows in addition an increase of tri- and monosialotransferrin. The aims of diploma thesis were: 1) to evaluate reference range for spectrum of sialylated forms of Tf separated by IEF and 2) to perform biochemical and molecular analyses in three patients (P1-P3) with clinical suspicion for CDG. Serum and genomic DNA from three patients with clinical suspicion for CDG and family members of P1 were analysed. Sera from 99 healthy volunteers within the age range of 2-42 years served as a control group. Tf was analysed by IEF with direct immunofixation, SDS-PAGE and Western blot using specific antibody against human Tf (Dako). Profiles of Tf were quantified by AlphaEaseFC software (Alpha Innotech). Data were analysed by software STATISTICA 9.0 (StatSoft). TF a PMM2 genes were analysed...

The goal of this project is to eliminate electroosmotic flow (EOF) in a microfluidic chip. EOF is a naturally occurring phenomenon at the fluid-surface interface in microfluidic chips when an electric field is applied across the fluid. When isoelectric focusing (IEF) is carried out to separate proteins based on their surface charge, the analytes must remain in the separation chamber, and not migrate to adjacent features in the microfluidic chip, which happens with EOF. For this project, a microfluidic chip was designed and commissioned to be photolithographically transferred onto a Si wafer. A PDMS component was then casted on the Si wafer and plasma bonded to a glass substrate. This chip was initially designed to carry out continuous IEF, and the focus of the project was shifted to the analysis of eliminating EOF in a microfluidic chamber. Per previous research test methods, methylcellulose will be used to analyze the phenomenon of electroosmotic flow in the chamber. A COMSOL model is used a theoretical basis of comparison when analyzing the flow velocities of the treated versus untreated microfluidic chips. The purpose of this project is to use the research performed in on this chip as a precursor to future analyses of continuous IEF on microfluidic chips in the Cal Poly Microfluidics group.

Thesis (PhD)--Macquarie University, Division of Environmental & Life Sciences, Dept. of Chemistry & Biomolecular Sciences, 2009. / Bibliography: p. 200-212. / Introduction -- Shotgun proteomic analysis of rat liver membrane proteins -- A combination of immobilised pH gradients improve membrane proteomics -- Affects of tumor-induced inflammation on membrane proteins abundance in the mouse liver -- Affects of tumor-induced inflammation on biochemical pathways in the mouse liver -- General discussion -- References. / The aim of this thesis was to develop a proteomics methodology that improves the identification of membrane proteomes from mammalian liver. Shotgun proteomics is a method that allows the analysis of proteins from cells, tissues and organs and provides comprehensive characterisation of proteomes of interest. The method developed in this thesis uses separation of peptides from trypsin digested membrane proteins by immobilised pH gradient isoelectric focusing (IPG-IEF) as the first dimension of two dimensional shotgun proteomics. In this thesis, peptide IPG-IEF was shown to be a highly reproducible, high resolution analytical separation that provided the identification of over 4,000 individual protein identifications from rat liver membrane samples. Furthermore, this shotgun proteomics strategy provided the identification of approximately 1,100 integral membrane proteins from the rat liver. The advantages of using peptide IPG-IEF as a shotgun proteomics separation dimension in conjunction with label-free quantification was applied to a biological question: namely, does the presence of a spatially unrelated benign tumor affect the abundance of mouse liver proteins. IPG-IEF shotgun proteomics provided comprehensive coverage of the mouse liver membrane proteome with 1,569 quantified proteins. In addition, the presence of an Englebreth-Holm-Swarm sarcoma induced changes in abundance of proteins in the mouse liver, including many integral membrane proteins. Changes in the abundance of liver proteins was observed in key liver metabolic processes such as fatty acid metabolism, fatty acid transport, xenobiotic metabolism and clearance. These results provide compelling evidence that the developed shotgun proteomics methodology allows for the comprehensive analysis of mammalian liver membrane proteins and detailed some of the underlying changes in liver metabolism induced by the presence of a tumor. This model may reflect changes that could occur in the livers of cancer patients and has implications for drug treatments. / Mode of access: World Wide Web. / 609 p. ill. (some col.)

Master of Science / Department of Chemistry / Christopher T. Culbertson / Capillary isoelectric focusing (cIEF) is one of several electrophoretic separation techniques for proteins and various other bio - molecules widely used in biochemistry laboratories. A wide range of analytes separable by the different modes of Capillary Electrophoresis (CE) includes from a small organic or an inorganic molecule to the complex bio-molecules such as protein, peptides, cell organelles, and live microorganisms (e.g. bacteria and viruses). Of the various modes of electrophoresis, Isoelectric focusing (IEF) is a good method for the separation of large amphoteric molecules such as peptides and proteins because of the attainment of overall surface charge depending up on its environment pH. This thesis mainly focuses on application of cIEF for proteins separation and viruses’ detection, which is one of the biggest concerns of human and animal health because of viral outbreak causing loss of thousands of lives and property every year. In chapter one of this thesis, the principles and mechanisms of separation of CE, cIEF, comparative advantages of dynamic coatings over static coating, and advantages of Whole Column Imaging Detection (WCID) over On - olumn Single Point Detection have been discussed. Chapter two includes experimental procedure and calculations for EOF determination. The results of cIEF experiments with standard proteins to develop calibration curve followed by UV absorbance detection of two bacteriophage viruses TR4 and T1 are presented in the chapter three. Final chapter four includes the conclusion and discussion on future direction for the project. The main motivation for this work was to develop a method which is less labor intensive and requires shorter detection time compared to traditional detection methods such as virus culture in serology (7days), polymerase chain reaction (PCR) and gel electrophoresis (6hrs to 2days). A commercially available dynamic coating reagent, EoTrol LN® copolymer used our CE experiments found to be more convenient and efficient than commonly used surface modifiers for example silane-based reagents. Preliminary determination of the pIs of these T1 and TR4 by cIEF was 3.1 ± 1.0 and 6.8 ± 1.0 respectively. The pI of viruses can differ by their strains and the phase of virus - growth. The viruses, though closely related, are easily distinguishable by their different pIs.

Analysis of low-abundance components in small samples remains a challenge within bioanalytical chemistry, and new techniques for sample pretreatments followed by sensitive and informative detection are required. In this thesis, procedures for preconcentration and separation of proteins and peptides in open microchannels fabricated on silicon microchips are presented. Analyte electromigration was induced by applying a voltage along the channel length, and detection was performed either by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) within the open channel, or by sampling a nL fraction containing the preconcentrated analytes from the channel for subsequent nano-electrospray ionization- (nESI-) or MALDI-MS. Utilizing solvent evaporation from the open system during sample supply, sample volumes exceeding the 25-75 nL channel volume could be analyzed. For preconcentration/separation of components in the discrete channel volume a lid of inert fluorocarbon liquid was used for evaporation control. In Papers I and II, aqueous, carrier-free solutions of proteins and peptides were analyzed, and the method was successfully applied for fast and simple preconcentration of amyloid beta (Aβ) peptides, related to Alzheimer’s disease. The impact of possible impurities in the analysis of carrier-free solutions was investigated in Paper III with the 1D simulation software GENTRANS, and a method for open-channel isoelectric focusing in a tailor-made pH gradient was developed. The latter approach was used in Paper IV for preconcentration and purification of Aβ peptides after immunoprecipitation from cerebrospinal fluid and blood plasma, followed by MALDI-MS from a micropillar chip. Paper V includes simulations of an isotachophoretic strategy for selective enrichment of Aβ peptides. GENTRANS simulations were used to select the electrolyte composition, and 2D simulations in a geometry suitable for on-chip implementation were performed using COMSOL Multiphysics. / <p>QC 20160930</p>

submitted by Liu Dan. / Thesis (M.Sc.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 66-80). / Abstract also in Chinese. / CONTENTS --- p.i / LIST OF TABLES --- p.iii / LIST OF FIGURES --- p.iv / LIST OF ABBREVIATIONS --- p.v / ACKNOWLEDGEMENTS --- p.vi / ABSTRACT --- p.vii / Chapter Chapter 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- History --- p.1 / Chapter 1.2 --- Immunoglobulins --- p.3 / Chapter 1.2.1 --- Structure --- p.3 / Chapter 1.2.2 --- Properties of immunoglobulins --- p.7 / Chapter 1.3 --- Monoclonal proteins and monoclonal gammopathies --- p.12 / Chapter 1.3.1 --- Monoclonal proteins --- p.12 / Chapter 1.3.2 --- Monoclonal gammopathies --- p.14 / Chapter 1.4 --- Laboratory investigation of monoclonal immunoglobulin --- p.17 / Chapter 1.4.1 --- The current procedure of investigation in laboratory --- p.17 / Chapter 1.4.2 --- Problems in identifying monoclonal immunolgobuin --- p.19 / Chapter 1.5 --- Comparison of different techniques --- p.20 / Chapter 1.5.1 --- Immunoelectrophoresis --- p.20 / Chapter 1.5.2 --- Immunofixation electrophoresis --- p.22 / Chapter 1.5.3 --- Isoelectric focusing and immunoisoelectric focusing --- p.24 / Chapter 1.6 --- Aim of the present study --- p.27 / Chapter 1.7 --- Design of experiment --- p.27 / Chapter Chapter 2 --- MATERIALS AND METHODS --- p.30 / Chapter 2.1 --- Study subjects --- p.30 / Chapter 2.2 --- Apparatus --- p.30 / Chapter 2.2 --- Apparatus --- p.30 / Chapter 2.3 --- Reagents and materials --- p.32 / Chapter 2.4 --- Preparation of gels --- p.35 / Chapter 2.5 --- Isoelectric focusing procedure --- p.36 / Chapter 2.6 --- Acid fixation and staining --- p.37 / Chapter 2.7 --- Technical factors affecting results --- p.38 / Chapter Chapter 3 --- RESULTS --- p.40 / Chapter 3.1 --- Interpretation of results in isoelectric focusing --- p.40 / Chapter 3.2 --- Affecting factors --- p.47 / Chapter 3.3 --- Comparison of the results between IEF and IFE --- p.53 / Chapter Chapter 4 --- DISCUSSION --- p.59 / Chapter Chapter 5 --- CONCLUSION --- p.65 / References --- p.66

Ein Signalweg während der neuronalen Entwicklung im adulten Gehirn ist der PI3K-PTEN-Akt Signalweg. Akt ist eine Kinase die drei verschiedene Isoformen besitzt, welche durch die Phosphorylierung von S473 und T308 aktiviert werden. KO Modelle der Isoformen haben gezeigt, dass nicht alle Funktionen von anderen Isoformen kompensiert werden können. Die genaue Rolle der einzelnen Isoformen in einem neuronalen Zusammenhang ist nur wenig untersucht. Ziel dieser Arbeit war, eine detaillierte Analyse der einzelnen Akt Isoformen nach der Aktivierung des PI3K-PTEN Signalweges. Dazu wurde im Labor eine neue Methode zur isoelektrischen Fokussierung etabliert., welche Proteine nach ihrer Ladung trennt und somit eine Analyse der Dynamik von Akt Phosphorylierungen in neuronalen Zellen erlaubt. Im Zuge dieser Arbeit konnten wir bisher unerkannte Merkmale der Akt Aktivierung und Phosphorylierung identifizieren. Wir konnten zeigen, dass die S473 und T308 Phosphorylierung in Neuroblastomazellen unabhängig voneinander auftreten kann und, dass verschiedene Akt1 Moleküle unterschiedlich auf die Inhibition von PI3K reagieren. Außerdem konnten wir Verschiebungen in der Aktivierung und in der Expression der unterschiedlichen Isoformen während der postnatalen Gehirnentwicklung der Ratte feststellen. Des Weiteren konnten wir zeigen, dass die Aktivierung von Akt von dem Signal und dem Alter der Neurone abhängig ist. Noch nicht vollständig differenzierte Neurone reagieren vor allem auf BDNF Stimulation, wohingegen adulte, differenzierte Neurone hauptsächlich auf EGF reagieren und dort explizit Akt2 über EGFR und PI3K-p110α Signale aktiviert wird. Im Gegensatz dazu führt der Verlust von PTEN zu einer Aktivierung von hauptsächlich Akt1. Zusammenfassend zeigt diese Arbeit einen komplexen Zusammenhang der Phosphorylierung von Akt auf, welcher Signal- und Entwicklungsabhängig ist bei dem unterschiedliche Akt Populationen auf Wachstumsfaktoren und auf PTEN Verlust reagieren. / A major pathway involved in neuronal development is the PI3K-PTEN-Akt pathway. Akt comprises three isoforms, which are activated by phosphorylation of the residues S473 and T308. KO animals for the isoforms have shown differential as well as redundant functions of the three isoforms. However, their individual role in neuronal signaling pathways has not yet been studied in great detail. The aim of this study was to obtain further insight into differential Akt isoform signaling in response to changes in the activity of PI3K and PTEN pathway. A new isoelectric focusing method was established, which allowed us to separate Akt proteins according to their charge, therefore, providing a refined read-out to study dynamics of Akt phosphorylation in a neuronal background. In the course of this project we were able to identify previously undescribed features of Akt phosphorylation and activation. First, we could provide evidence for an uncoupling of the two activating phosphorylation events at S473 and T308 in neuroblastoma cells and differential sensitivities of Akt1 forms towards PI3K inhibition. Secondly, we found a transient shift in Akt isoform activation and abundance during postnatal rat brain development. Thirdly, we were able to show that the activation of different Akt isoforms is dependent of the upstream signal as well as the age of the neuron. Immature neurons were found to be highly responsive to BDNF treatment, whereas mature neurons were most responsive to EGF stimulation leading exclusively to activation of Akt2 in an EGFR- and PI3K/p110α-dependent manner. Stimulation of Akt phosphorylation by the loss of PTEN led to an activation of mainly Akt1 forms, which suggests inherent differences in the Akt pools that are accessible to growth factors dependent PI3Ks as compared to the pools that are controlled by PTEN. In summary, this thesis demonstrates the presence of complex phosphorylation events of Akt in a developmental- and signal-dependent manner in neurons.