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Poster presentation

Pro-Detect Rapid Assay Lateral Flow Kits for detecting protein tags and antibody isotyping

Krishna Vattem, PhD

Speaker:

Krishna Vattem, PhD
Senior R&D Scientist, Protein and Cell Analysis, Thermo Fisher Scientific

Biography:

Krishna’s Ph.D. and postdoctoral work focused on understanding control mechanisms regulating the initiation of protein translation in eukaryotes. Following his academic training, he joined Thermo Fisher Scientific and has led multiple projects including the development of the HeLa and CHO lysate based cell-free translation systems, also known as  Thermo Scientific 1-Step In Vitro Translation (IVT) Systems, which contain all the necessary reagents to express proteins of interest in as little as one hour.

Abstract

Lateral flow assays (LFAs) are rapid, sensitive, and easy-to-use immunoassays for detecting specific target proteins in vitro. Although the LFA format is extremely popular in point-of-care diagnostic tests (i.e., pregnancy tests), to-date its use in basic research laboratories is not widespread. Here we present the Thermo Scientific Pro-Detect Rapid Assay, an LFA designed for research workflows that can be used for rapid monitoring of expressed recombinant proteins and rapid identification of antibody isotypes. The Pro-Detect Rapid Assay is an alternative to western blotting and ELISA formats that require significantly more time for sample processing as well as additional instrumentation. The rapid qualitative (yes/no) determination of this LFA will also help address key pain points in cell culture and protein expression workflows.

Learning Objectives

  • Understand how lateral flow technology–based Pro-Detect Rapid Assays can be used to rapidly detect protein expression
  • Understand how lateral flow technology–based Pro-Detect Rapid Assays can be used to rapidly isotype human, mouse, or rat antibodies

Webinar

High throughput production of autoantigens for rapid detection of multiple autoimmune diseases

Aimin Xu, PhD

Speaker:

Aimin Xu, PhD
Professor in Medicine, Professor in Pharmacology and Pharmacy, Director of State Key Laboratory of Pharmaceutical Biotechnology, at the University of Hong Kong

Biography:

Dr. Aimin Xu is currently a chair professor for the Department of Medicine and the Department of Pharmacology & Pharmacy and the director of the State Key Laboratory of Pharmaceutical Biotechnology at the University of Hong Kong. His major research interest is the discovery and functional characterization of novel hormones and biomarkers involved in the pathogenesis of metabolic and autoimmune diseases. His team has identified the glycol-isoforms of adiponectin and the FGF21-adiponectin axis in protection against obesity-related cardiometabolic syndrome. His team also discovered adipocyte fatty acid binding protein (A-FABP) and lipocalin-2 as pro-inflammatory adipokines in both rodents and humans. In addition, his work contributed significantly to the understanding of the molecular basis of adipose tissue inflammation and immunometabolic regulators in both metabolic and autoimmune diseases. His team has developed a series of immunoassays that are now widely used for clinical diagnostics, drug screening, and clinical and basic research for metabolic and immunological disorders.

Abstract

Current methods for in vitro diagnosis of autoimmune diseases (ADs) are mainly based on the detection of circulating autoantibodies using specific AD autoantigens. A high-quality recombinant autoantigen with epitopes resembling its natural form in the human body is the key to sensitive, specific, and rapid diagnosis. In this webinar we describe the use of the Gibco Bac-to-Bac Baculovirus Expression System to express 22 recombinant autoantigens in Spodoptera frugiperda 9 (Sf9) cells, with folding and posttranslational modifications matching those of the natural autoantigens in vivo. The Bac-to-Bac system allowed us to generate recombinant baculoviruses expressing the target genes via transposition within 48 hours. Using Gibco Sf-900 II SFM, we were able to propagate Sf9 cells to a density of approximately 6–8 x 106 cells/mL in a suspension culture and over 1–2 x 107 cells/mL during fermentation. These tools significantly reduce the time and labor costs needed for virus generation, amplification, and protein production, and allow high-throughput production of autoantigens. Using these 22 high-quality recombinant autoantigens, we have successfully developed a membrane-based chip for simultaneous screening of 7 major ADs that were validated in over 200 clinical samples. In addition to demonstrating greater than 95% sensitivity, the new assay procedure is very rapid (only taking 30 minutes).

Learning Objectives

  • Understand the advantages of the Bac-to-Bac Baculovirus Expression System
  • Demonstrate how the Bac-to-Bac Baculovirus Expression System can be used for high throughput production of high-quality recombinant autoantigens

Poster presentation

High throughput proteomic applications using anti-DYKDDDDK magnetic agarose

Betsy Benton, PhD

Speaker:

Betsy Benton, PhD
R&D Staff Scientist, Protein and Cell Analysis, Thermo Fisher Scientific

Biography:

Betsy Benton completed her PhD and postdoctoral research at the University of Wisconsin-Madison in the fields of biochemistry and human oncology. She joined Thermo Fisher Scientific in 2000, where she has developed numerous products in the areas of protein purification (with both magnetic beads and resin supports), protein sample preparation, and protein detection.

Abstract

Affinity purification, immunoprecipitation, and mass spectrometry analysis are essential tools used in proteomic studies. A successful outcome is facilitated by high-capacity supports, efficient sample preparation, and reproducibility provided by automation. We demonstrate that an anti-DYKDDDDK magnetic agarose fulfills these criteria. The DYKDDDDK epitope tag is commonly used in protein expression systems as a short purification handle. Using an antibody against a DYKDDDDK tag covalently attached to high-binding capacity magnetic agarose, we demonstrate effective purification of recombinant proteins expressed in bacteria, the Invitrogen ExpiCHO Expression System, and by a HeLa in vitro translation (cell-free) system. Biological activity was demonstrated for ExpiCHO-expressed DYKDDDDK–Tumor Necrosis Factor alpha (TNFα) which was used to successfully stimulate cells and activate Nuclear Factor kappa B (NFkB) p65. In addition, successful co-immunoprecipitation of DYKDDDDK-tagged BAD with 14-3-3 proteins was confirmed using an optimized sample preparation protocol in conjunction with the Thermo Scientific KingFisher Flex Magnetic Particle Processor and mass spectrometry. In summary, anti-DYKDDDDK magnetic agarose can be used to effectively isolate, purify, and enrich functional protein targets in a variety of proteomic applications.

Learning Objectives

  • Learn how to automate protein applications using cell-based and cell-free systems with magnetic beads

Webinar

Importance of sample preparation and choosing the right gel for SDS-PAGE

Alok Tomar, PhD

Speaker:

Alok Tomar, PhD
Product Manager, Protein and Cell Analysis, Thermo Fisher Scientific

Biography:

Alok’s PhD and postdoctoral work focused on understanding the role of cytoskeleton and focal adhesion complexes on cell migration and metastasis of cancer cells, resulting in 19 publications in leading peer-reviewed journals. He has been working as a product management professional for over 6 years, launching several next-generation sequencing (NGS)-based genetic research tests that can assess risks of developing cancer. Currently he is a product manager for the Thermo Fisher Scientific protein gel electrophoresis portfolio and has recently launched new Invitrogen precast gels that provide reproducible quality and performance.

Abstract

To achieve clear and accurate resolution of protein bands in protein gel electrophoresis, it is important to use the appropriate sample preparation conditions and to choose the right gel chemistry for a specific research application. Protein samples that are used for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) are often complex in nature and can include a wide range of contaminants such as nucleotides, lipids, excess salts and reducing agents, and insoluble material, to name a few. The mistakes that are commonly made during sample preparation also include over-heating samples (temperature and time), using incorrect reducing agents, incorrect protein-to-sample buffer ratio, insoluble material in the sample, and over- and under-loading of protein sample.

Similarly, choosing the right gel chemistry for a specific research application can help minimize the validation time for that particular application. For example, neutral pH Bis-Tris gels are recommended for samples with low abundance of target proteins or when downstream applications require high protein integrity (mass spectrometry, posttranslational modification, or protein sequencing). Additionally, high molecular weight proteins (up to 500 kDa) can be optimally resolved by using Tris-acetate gels, whereas low molecular weight proteins (as low as 2.5 kDa) can be optimally resolved using Tricine gels. In this webinar, we will provide an overview of your options and guidance on how to get optimal protein resolution.

Learning objectives

  • Importance of sample preparation in SDS-PAGE electrophoresis
  • Importance of choosing the right gel chemistry for specific research applications

Webinar

Light up your western blots – fluorescent western blotting tips, tricks & more

paul-haney

Speaker:

Paul Haney, Ph.D.,
Senior Product Manager, Protein and Cell Analysis, Thermo Fisher Scientific

Biography:

Over the past 18 years, Paul Haney has been a key member of the R&D and product management team in the protein biology business unit of Thermo Fisher Scientific, where he has provided leadership and strategy for the development of products for the life science research market. He is focused on products for western blot detection, western blot and gel imaging, protein sample preparation, protein isolation, immunodetection, and mass spectrometry analysis. Before joining Thermo Fisher Scientific, Paul conducted postdoctorial research in receptor biology at the Mayo Clinic and in bioinformatics and protein stability at the University of Illinois/Urbana. Paul completed his PhD in molecular biology and protein biology at the University of Illinois/Urbana.

Abstract

While chemiluminescence has been the traditional approach to western blot detection, this strategy is being progressively supplemented or replaced with fluorescence-based detection. With the introduction of advanced CCD-camera–based imaging systems and scanning instruments, along with improvements in fluorescent conjugate technologies, scientists now have the necessary tools available to take advantage of the range of fluorescent dyes and antibodies for fluorescent western blot detection. Learn more about the best practices to help enable you to become successful at fluorescent western blotting.

Learning objectives

  • Understand the fundamentals of fluorescent western blotting
  • Successfully design fluorescent western blotting experiments

Poster presentation

Alternative blotting methods

Boguslawa “Bea” Dworecki, MS

Speaker:

Boguslawa “Bea” Dworecki, MS
Research Scientist, Protein and Cell Analysis, Thermo Fisher Scientific

Biography:

Bea is an R&D scientist in the protein analysis group. She completed her BS at Rockford University in 1997 in chemistry. She obtained her MS in medical biotechnology from the University of Illinois College of Medicine at Rockford in 2010. Over the past 20 years, she has worked on the development of numerous products for the life science research market. She has extensive experience in electrophoresis, protein transfer and detection, western blot and gel imaging, protein sample preparation, fluorescent labeling and purification of proteins and antibodies, dialysis, and protein staining and transfer devices.

Abstract

Western blotting is an important technique for characterizing specific protein targets. Successful western detection is dependent on the quality of protein transfer from polyacrylamide gels to the blotting membranes, and on the processing steps and reagents used. The traditional wet transfer method offers high transfer efficiency, but at the cost of time and effort. Improvements to dry and semi-dry transfer systems have greatly improved the transfer speed (less than 10 min) and convenience without compromising transfer efficiency. This poster will discuss the traditional wet blotting transfer technique as compared to dry and semi-dry transfer techniques.

Learning objectives

  • Compare the classical wet western blotting technique to dry and semi-dry blotting techniques
  • Learn about dry and semi-dry western blotting for high-efficiency transfer and quality results

Webinar

Anchored Protein Kinase-A (PKA) as a gatekeeper for the Connexin-43 gap junction

Guillaume Pidoux, PhD

Speaker:

Guillaume Pidoux, PhD
Senior Researcher at Inserm UMR-S 1180 (CRCN)

Biography:

Guillaume Pidoux completed his PhD at University Paris Descartes (Paris, France) in 2006 and did a postdoctoral fellowship at the Nordic EMBL, NCMM—University of Oslo (Oslo, Norway) in 2010. In 2012, he was recruited as a senior researcher at Inserm (French National Institute for Health and Medical Research). He is interested in the spatiotemporal regulation of cAMP signaling in physiological and pathophysiological conditions. He has recently established a small group at Inserm UMR-S 1180, which focuses on understanding the direct role of A-Kinase Anchoring Proteins (AKAPs) and Protein Kinase-A (PKA) in cardiac physiology and during the development of cardiomyopathies. With his cell biology and biochemistry approaches, he is paving the way to develop new therapeutic schemes aimed at improving the quality of life for patients.

Abstract

Anchored Protein Kinase-A (PKA) bound to A-Kinase Anchoring Protein (AKAP) mediates the effects of localized increases in cAMP in defined subcellular microdomains and retains the specificity in cAMP–PKA signaling to distinct extracellular stimuli. Gap junctions are pores between adjacent cells constituted by connexin (Cxs) proteins that provide a means of communication, electrical impulse propagation, and transfer of small molecules. One of the most widely and highly expressed Cxs is Cx43, which was shown to be regulated through phosphorylation by several kinases including PKA. Ezrin is a membrane-associated protein that can serve as an AKAP and hold an anchored pool of PKA. We found PKA to be part of a macromolecular signaling complex with ezrin and gap junction protein Cx43 that provides cAMP-mediated control of gap junction communication. We established that an inhibition of PKA activity or disruption of the ezrin–Cx43 interaction abolished PKA-dependent phosphorylation of Cx43 as well as gap junction communication. In vitro studies using peptide arrays, together with mass spectrometry, pointed to serine 369 and 373 of Cx43 as the major PKA phosphorylation sites. A combination of knockdown and reconstitution experiments and gap-FLIP assays with mutant Cx43 containing single or double phosphoserine–mimicking amino acid substitutions in putative PKA phosphorylation sites demonstrated that phosphorylation of S369 and S373 mediated gap junction communication. We propose that the PKA–ezrin–Cx43 macromolecular complex regulating gap junction communication constitutes a general mechanism to control opening of Cx43 gap junctions by phosphorylation in response to cAMP signaling in various cell types (i.e., heart, liver, and placenta).


Webinar

Complete and innovative western workflow solutions: filling the gap between art and science (presented in Chinese)

Jiao Yang, PhD

Speaker:

Jiao Yang, PhD
Technical Application Specialist at Thermo Fisher Scientific

Biography:

Jiao Yang works as a technical application specialist at Thermo Fisher Scientific in Shanghai, China. She received her PhD in biochemistry and molecular biology from Shanghai Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences. She has been working on the mechanism of protein degradation for about six years and has published a paper in cell research as a first-time author. In 2016, she joined Thermo Fisher Scientific and supports products in protein biology.

Abstract

Western blotting is a widely used and accepted technique to detect levels of protein expression in a cell or tissue extract. This webinar will cover the electrophoresis, transfer, and detection steps of the western blotting workflow, highlight our popular products, and introduce tips and tricks for superior western analysis to make western blotting more efficient and effective.

Learning objectives

  • Overview of the critical steps in protocols for western blot, electrophoresis, protein transfer, and western detection
  • Learn about our total western blotting solutions
  • Tips and tricks for superior western blot analysis

Poster presentation

Rapid Biuret based colorimetric assay for protein quantitation

Ramesh Ganapathy, PhD

Speaker:

Ramesh Ganapathy, PhD
R&D Scientist III, Protein and Cell Analysis, Thermo Fisher Scientific

Biography:

Ramesh Ganapathy joined Thermo Fisher Scientific in 2001 after completing his PhD at the University of Wisconsin-Madison and a postdoctoral fellowship at the Department of Bioengineering at the University of Washington-Seattle. He also holds his MBA degree from Northern Illinois University. Since joining Thermo Fisher Scientific, he has worked on developing a broad range of products including resins, substrates for western blotting, magnetic beads, and peptide and protein assay products.

Abstract

Protein quantitation assays are essential for workflows that include protein extraction, labeling, and analysis. The simplest method for protein quantitation is measuring the absorbance of a protein sample at 280 nm (A280) and calculating the quantity using the Beer Lambert Law. However, this method is only applicable when the sample is a purified protein solution. Most cell biology experiments result in mixed or unpurified protein samples extracted from cell or tissue lysates. The Bradford assay (or Coomassie dye–based assay) has been a standard protein quantitation assay for unpurified proteins for some time, but a major drawback of this assay is its poor linearity. An improved assay, the Thermo Scientific Pierce BCA Protein Assay using bicinchoninic acid, is a chelator-based assay with exceptional linearity and low protein-to-protein variability that was quickly established as the “gold standard” for protein assays. One drawback with this assay, though, is the relatively long incubation time (30 minutes at 37˚C or 2 hours at room temperature). This poster presents the development of an improved BCA protein quantitation assay, the new Thermo Scientific Pierce Rapid Gold BCA Protein Assay, that addresses the need for a shorter incubation time (5 minutes at room temperature). Herein we present our evaluation of protein-to-protein variability, accuracy of “known” protein mixes, and closeness in protein estimation for this new assay as compared to the classic BCA assay. The results of this evaluation demonstrate that the new Pierce Rapid Gold BCA assay with the shorter incubation time provides the same accuracy as the original BCA assay.

Learning objectives:

  • A rapid way to quantitate protein using the same principle as the traditional BCA assay

Webinar

Protein sample evaluation using the NanoDrop One UV-Vis Spectrophotometer

Voula Kodoyianni, PhD

Speaker:

Voula Kodoyianni, PhD
Product Marketing Manager—NanoDrop instruments, Thermo Fisher ScientificThermo Fisher Scientific

Biography:

Voula Kodoyianni is the product manager for Thermo Scientific NanoDrop microvolume UV-Vis instruments and led the development and market launch of the NanoDrop One UV-Vis Spectrophotometer in 2015. Prior to joining Thermo Fisher Scientific, Dr. Kodoyianni was the chief scientific officer for GWC Technologies, led a genome sequencing team at the University of Wisconsin-Madison, and was a Howard Hughes Medical Institute Fellow conducting research in C. elegans development. She holds a master’s degree in chemistry from Duke University and a PhD in biochemistry from the University of Wisconsin-Madison.

Abstract

Protein sample evaluation is an important step in many protein workflows. The NanoDrop One microvolume spectrophotometer supports protein sample quantification with multiple preconfigured applications, including direct absorbance at A280, A205, or other wavelengths using the Thermo Scientific Pierce colorimetric assays (BCA, Rapid Gold BCA, Pierce 660, Bradford, etc.) and a proteins and labels application for quantitation of metalloproteins and fluorescently labeled proteins. We will review what you need to consider when choosing a protein quantification method for your sample. Is the sample one purified protein or a mixture of proteins? Does your peptide contain tyrosine and tryptophan residues? Does the sample buffer absorb in the UV spectrum? How can co-purified DNA affect your protein sample measurement? What is a good blank/control for your sample? Answers to these questions and other key considerations will be discussed.

Learning objectives:

  • Understand protein extinction coefficients—how to obtain the most accurate protein sample quantification using UV absorbance at 280 nm
  • Learn how new Thermo Scientific Acclaro sample intelligence technology in the NanoDrop One spectrophotometer delivers information not only about the concentration but also the quality of your protein sample

Poster presentation

Development of Multiplex xMAP Technology-Based Assays for Simultaneous Detection of Soluble Checkpoint Molecules Involved in Anti-Cancer Immune Response

Susanne Oehler, PhD

Speaker:

Susanne Oehler, PhD
Senior Manager, Research and Development, Antibodies and Immunoassays, Thermo Fisher Scientific

Biography:

Susanne received her PhD at the University of Vienna where she characterized the gene of a major cytoskeletal interlinking protein, plectin, which affects dynamic properties of the cytoskeleton and plays a role in tumor biology. Her postgraduate work focusing on chemotherapy resistance mechanisms in breast cancer cells was carried out at the Ludwig Bolzmann Institute of Clinical Experimental Oncology at the General Hospital in Vienna. Currently Suzanne is a senior scientist in the antibodies and immunoassays group in R&D at Thermo Fisher Scientific. She is a group leader in the multiplex immunoassays development team.

Abstract

To ensure the immune system’s balance between the recognition of non-self and the prevention of autoimmunity, the activity of immune cells needs to be strictly controlled. During the process of carcinogenesis, tumor cells try to resist immune responses and work to suppress the cycle of cancer immunity through multiple mechanisms involving regulatory proteins. These proteins—referred to as immune checkpoints—can affect immunoregulatory pathways by either boosting (co-stimulatory) or restricting (co-inhibitory) the immune response. For the majority of these checkpoint markers, soluble isoforms or shed variants that function as immune adjuvants or decoy receptors, have been identified. To date, cancer therapies that use antibodies targeted to the proteins within these immune checkpoint pathways have proven effective. It is also hypothesized that the measurement of the circulating concentration of the soluble forms of the immune checkpoint proteins could potentially correlate with the clinical efficacy of corresponding checkpoint modulator drugs.

To test this hypothesis, we developed two comprehensive multiplexing panels (using Luminex® xMAP® technology) that are specifically designed to detect soluble checkpoint molecules involved in T cell and NK cell regulation. The panels allow the simultaneous detection of immune stimulatory and inhibitory factors, allowing a more holistic picture of the molecular players in cancer immunity. Preliminary results using the Invitrogen Immuno-Oncology Checkpoint 14-Plex Human ProcartaPlex Panel 1 (Cat. No. EPX14A-15803-901) and the Invitrogen Immuno-Oncology Checkpoint 14-Plex Human ProcartaPlex Panel 2 (Cat. No. EPX140-15815-901) demonstrate that there are different expression patterns for each that are dependent on both disease state and patient treatment. More in-depth analysis of these soluble biomarkers will hopefully shed more light on the biology of the immune checkpoint pathways and provide a possible tool for monitoring response to therapeutic treatment.

Learning objectives:

  • Investigate the advantages of using soluble checkpoint molecules as potential markers for the response to chemotherapy
  • Develop immunoassays for new immune regulating targets

Webinar

Invitrogen immunoassays: vast choice of appropriate tools for specific needs

Markus Miholits, MS

Speaker:

Markus Miholits, MS
Senior Manager, Research and Development, Antibodies and Immunoassays, Thermo Fisher Scientific

Biography:

Markus leads the R&D group at the Vienna Center of Excellence for Immunoassay Development at Thermo Fisher Scientific. He is responsible for product and technology development as well as contract and custom lab services. Prior to joining Thermo Fisher Scientific, Markus was head of the laboratory for immunoassay development at Affymetrix and was group leader for immunoassay development at eBioscience. Also, he was project leader of R&D for immunoassay development and establishment of new immunoassay technologies (instant ELISA assay; in-house developed, bead-based multiplexing immunoassay technology) at Bender MedSystems GmbH. Markus holds a master’s degree from the University of Vienna (Institute of Biochemistry).

Abstract

Understanding the complex, yet coordinated protein interactions that sustain life, may help unlock the mysteries of disease mechanisms and thereby treatments. Protein identification and quantification have become extremely valuable in advancing our understanding and finding cures. As there are many methodologies available to scientists today, it can be challenging to choose which tools are the most effective to answer specific questions.

Based on the decades of market experience in the immunological research reagents market, Thermo Fisher Scientific addresses different customer needs by different technologies for detecting the same analytes and offers a vast choice of appropriate tools for the customer’s specific needs.

Learning objectives:

  • Identify time-saving technologies and tools for protein quantitation
  • Understand the benefits of choosing from different immunoassay platforms and formats, all from the same provider

Webinar

ProQuantum High-Sensitivity Immunoassays offer minimum sample consumption With maximum performance

David Bourdon, PhD

Speaker:

David Bourdon, PhD
Senior R&D Manager, Antibodies and Immunoassays, Thermo Fisher Scientific

Biography:

David’s PhD and postdoctoral work focused on the study of G protein–coupled receptor signaling and thrombosis. Following his academic training, he led multiplexed immunoassay automation efforts at a Luminex partnering company. Joining Thermo Fisher Scientific in 2009, David is now a senior R&D manager focusing on next-generation immunoassay development including the Invitrogen ProQuantum platform. David and his team continue working with translational investigators to identify serum-based biomarkers in cancer, autoimmunity, and inflammation.

Abstract

Early detection of protein biomarkers is critical to the study of human diseases, yet current tools available for low-level protein quantitation do not address all the needs for protein and molecular biologists alike. A common concern is sample volume limitation due to difficulties in obtaining sufficient biological samples from various models in research studies. Additionally, cost and time-to-answer are always a factor when considering which assay platform to use. In many cases, end users must make trade-offs on which factors impact their work the least.

Invitrogen ProQuantum High-Sensitivity Immunoassays combine multiple benefits into a single platform so that users don’t need to make trade-offs. The platform works by combining the analyte specificity of high-affinity antibody–antigen binding with the signal detection and amplification of real-time PCR. The result is an assay that can measure low-expressing proteins (femtogram levels) while only consuming a fraction of sample volume (i.e.,1–2 microliters per sample) compared to other immunoassay methods. Best of all, it uses a streamlined workflow (2 hours) that is highly scalable and affordable on an open, widely used qPCR platform.

Learning objectives:

  • To understand the multiple benefits of ProQuantum High-Sensitivity Immunoassays and how the platform works
  • To understand how the kits are designed for a simplified workflow amenable to high-throughput applications

Webinar

Signaling pathways at your fingertips – multiplex IP to targeted mass spectrometry assay kits for AKT/mTOR pathway

Bhavin Patel, MD, MS

Speaker:

Bhavin Patel, MD, MS
Senior R&D Scientist, Protein and Cell Analysis, Thermo Fisher Scientific

Biography:

Bhavin Patel works as a senior R&D scientist in mass spectrometry reagents group at Thermo Fisher Scientific. Bhavin has an MD degree from the Maharaja Sayajirao University of Baroda in India, and an MS in biotechnology from the Georgetown University. Bhavin had led a protein biomarker assay group at NextGen Sciences and a proteomics/mass spectrometry group at Fox Chase Cancer Center before joining Thermo Fisher Scientific in 2013. Since joining Thermo Fisher Scientific, Bhavin has led the development of targeted mass spectrometry (MS) assay kits, new MS standards, protein sample preparation reagents, an antibody validation method for IP-MS, and reagents for quantitative proteomic analysis.

Abstract

Proteomics offers a deeper understanding of the protein events influencing cellular and biological function. However, identifying key regulated proteins and their implicated signaling pathways represents a major analytical challenge to researchers. This is due to the broad dynamic range of protein abundance in biological samples and the inherent limitations of the instrumentation and methodologies utilized to probe and detect minute target proteins in complex cellular environments. In this webinar, we discuss targeted mass spectrometry (MS)-based approaches and the various strategies designed to reduce sample complexity through improved sample preparation and enrichment. Focusing on proteins involved in the AKT/mTOR signaling pathway, we provide guidance toward targeted MS assay development and validation, including robustness, precision, and accuracy of these assays. Finally, we’ll examine results of multiplexed IP to targeted MS assays compared to existing immunoassay techniques.

Learning objectives:

  • Development and validation of immunoenrichment to mass spectrometry (IP-MS) assays
  • Accurate and precise quantitation of multiple AKT/mTOR pathway proteins by multiplex IP to targeted MS assays and comparison to exiting immunoassay techniques

Webinar

Addressing the challenges of bioconjugation for improved crosslinking and modification

Greg Hermanson

Speaker:

Greg Hermanson
Chief Technology Officer and Principal at Aurora Microarray Solutions, Inc. & President of Greg T. Hermanson, Inc.

Biography:

Greg Hermanson is the chief technology officer and principal at Aurora Microarray Solutions, Inc. and the president of Greg T. Hermanson, Inc., a bioscience consulting company. Greg has over three decades of experience in the development of life science research and diagnostic products, with particular expertise in protein chemistry, assay development, immobilization, and bioconjugation. He is well known as the author of Bioconjugate Techniques, (now in its third edition), which is an extensive manual on the methods of bioconjugation. Greg has had a significant impact on the bioscience fields as proven by nearly 27,500 citations of his publications and patents.

Abstract

Bioconjugation is a critical technique for creating many of the key reagents used in research and commercial applications. This webinar will cover in great detail the major reactions used to form bioconjugates as well as the functional groups which react with them. This includes identifying the principle target groups on proteins, oligonucleotides, glycans, and carbohydrates and matching them with the appropriate reactive groups for conjugation. The reactive groups covered will include those useful for amine conjugations, thiol reactions, carbonyl reactions, and for bioorthogonal coupling.

This webinar will examine the major products of these reactions, as well as any potential side reactions or competing hydrolysis that may occur, and any interfering substances that should be avoided. The goal of this webinar is to provide a solid understanding of these reactions and how to optimize them to create the best possible bioconjugates for the intended downstream applications.

Learning objectives

  • Learn about the major reactive groups used in bioconjugation and how to optimize each reaction
  • Learn to identify major products of each reaction and understand the effects of hydrolysis, side reactions, and interfering substances
  • Discuss tips and tricks for improved results

Poster presentation

Pro-Detect Rapid Assay Lateral Flow Kits for detecting protein tags and antibody isotyping

Krishna Vattem, PhD

Speaker:

Krishna Vattem, PhD
Senior R&D Scientist, Protein and Cell Analysis, Thermo Fisher Scientific

Biography:

Krishna’s Ph.D. and postdoctoral work focused on understanding control mechanisms regulating the initiation of protein translation in eukaryotes. Following his academic training, he joined Thermo Fisher Scientific and has led multiple projects including the development of the HeLa and CHO lysate based cell-free translation systems, also known as  Thermo Scientific 1-Step In Vitro Translation (IVT) Systems, which contain all the necessary reagents to express proteins of interest in as little as one hour.

Abstract

Lateral flow assays (LFAs) are rapid, sensitive, and easy-to-use immunoassays for detecting specific target proteins in vitro. Although the LFA format is extremely popular in point-of-care diagnostic tests (i.e., pregnancy tests), to-date its use in basic research laboratories is not widespread. Here we present the Thermo Scientific Pro-Detect Rapid Assay, an LFA designed for research workflows that can be used for rapid monitoring of expressed recombinant proteins and rapid identification of antibody isotypes. The Pro-Detect Rapid Assay is an alternative to western blotting and ELISA formats that require significantly more time for sample processing as well as additional instrumentation. The rapid qualitative (yes/no) determination of this LFA will also help address key pain points in cell culture and protein expression workflows.

Learning Objectives

  • Understand how lateral flow technology–based Pro-Detect Rapid Assays can be used to rapidly detect protein expression
  • Understand how lateral flow technology–based Pro-Detect Rapid Assays can be used to rapidly isotype human, mouse, or rat antibodies

Webinar

High throughput production of autoantigens for rapid detection of multiple autoimmune diseases

Aimin Xu, PhD

Speaker:

Aimin Xu, PhD
Professor in Medicine, Professor in Pharmacology and Pharmacy, Director of State Key Laboratory of Pharmaceutical Biotechnology, at the University of Hong Kong

Biography:

Dr. Aimin Xu is currently a chair professor for the Department of Medicine and the Department of Pharmacology & Pharmacy and the director of the State Key Laboratory of Pharmaceutical Biotechnology at the University of Hong Kong. His major research interest is the discovery and functional characterization of novel hormones and biomarkers involved in the pathogenesis of metabolic and autoimmune diseases. His team has identified the glycol-isoforms of adiponectin and the FGF21-adiponectin axis in protection against obesity-related cardiometabolic syndrome. His team also discovered adipocyte fatty acid binding protein (A-FABP) and lipocalin-2 as pro-inflammatory adipokines in both rodents and humans. In addition, his work contributed significantly to the understanding of the molecular basis of adipose tissue inflammation and immunometabolic regulators in both metabolic and autoimmune diseases. His team has developed a series of immunoassays that are now widely used for clinical diagnostics, drug screening, and clinical and basic research for metabolic and immunological disorders.

Abstract

Current methods for in vitro diagnosis of autoimmune diseases (ADs) are mainly based on the detection of circulating autoantibodies using specific AD autoantigens. A high-quality recombinant autoantigen with epitopes resembling its natural form in the human body is the key to sensitive, specific, and rapid diagnosis. In this webinar we describe the use of the Gibco Bac-to-Bac Baculovirus Expression System to express 22 recombinant autoantigens in Spodoptera frugiperda 9 (Sf9) cells, with folding and posttranslational modifications matching those of the natural autoantigens in vivo. The Bac-to-Bac system allowed us to generate recombinant baculoviruses expressing the target genes via transposition within 48 hours. Using Gibco Sf-900 II SFM, we were able to propagate Sf9 cells to a density of approximately 6–8 x 106 cells/mL in a suspension culture and over 1–2 x 107 cells/mL during fermentation. These tools significantly reduce the time and labor costs needed for virus generation, amplification, and protein production, and allow high-throughput production of autoantigens. Using these 22 high-quality recombinant autoantigens, we have successfully developed a membrane-based chip for simultaneous screening of 7 major ADs that were validated in over 200 clinical samples. In addition to demonstrating greater than 95% sensitivity, the new assay procedure is very rapid (only taking 30 minutes).

Learning Objectives

  • Understand the advantages of the Bac-to-Bac Baculovirus Expression System
  • Demonstrate how the Bac-to-Bac Baculovirus Expression System can be used for high throughput production of high-quality recombinant autoantigens

Poster presentation

High throughput proteomic applications using anti-DYKDDDDK magnetic agarose

Betsy Benton, PhD

Speaker:

Betsy Benton, PhD
R&D Staff Scientist, Protein and Cell Analysis, Thermo Fisher Scientific

Biography:

Betsy Benton completed her PhD and postdoctoral research at the University of Wisconsin-Madison in the fields of biochemistry and human oncology. She joined Thermo Fisher Scientific in 2000, where she has developed numerous products in the areas of protein purification (with both magnetic beads and resin supports), protein sample preparation, and protein detection.

Abstract

Affinity purification, immunoprecipitation, and mass spectrometry analysis are essential tools used in proteomic studies. A successful outcome is facilitated by high-capacity supports, efficient sample preparation, and reproducibility provided by automation. We demonstrate that an anti-DYKDDDDK magnetic agarose fulfills these criteria. The DYKDDDDK epitope tag is commonly used in protein expression systems as a short purification handle. Using an antibody against a DYKDDDDK tag covalently attached to high-binding capacity magnetic agarose, we demonstrate effective purification of recombinant proteins expressed in bacteria, the Invitrogen ExpiCHO Expression System, and by a HeLa in vitro translation (cell-free) system. Biological activity was demonstrated for ExpiCHO-expressed DYKDDDDK–Tumor Necrosis Factor alpha (TNFα) which was used to successfully stimulate cells and activate Nuclear Factor kappa B (NFkB) p65. In addition, successful co-immunoprecipitation of DYKDDDDK-tagged BAD with 14-3-3 proteins was confirmed using an optimized sample preparation protocol in conjunction with the Thermo Scientific KingFisher Flex Magnetic Particle Processor and mass spectrometry. In summary, anti-DYKDDDDK magnetic agarose can be used to effectively isolate, purify, and enrich functional protein targets in a variety of proteomic applications.

Learning Objectives

  • Learn how to automate protein applications using cell-based and cell-free systems with magnetic beads

Webinar

Importance of sample preparation and choosing the right gel for SDS-PAGE

Alok Tomar, PhD

Speaker:

Alok Tomar, PhD
Product Manager, Protein and Cell Analysis, Thermo Fisher Scientific

Biography:

Alok’s PhD and postdoctoral work focused on understanding the role of cytoskeleton and focal adhesion complexes on cell migration and metastasis of cancer cells, resulting in 19 publications in leading peer-reviewed journals. He has been working as a product management professional for over 6 years, launching several next-generation sequencing (NGS)-based genetic research tests that can assess risks of developing cancer. Currently he is a product manager for the Thermo Fisher Scientific protein gel electrophoresis portfolio and has recently launched new Invitrogen precast gels that provide reproducible quality and performance.

Abstract

To achieve clear and accurate resolution of protein bands in protein gel electrophoresis, it is important to use the appropriate sample preparation conditions and to choose the right gel chemistry for a specific research application. Protein samples that are used for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) are often complex in nature and can include a wide range of contaminants such as nucleotides, lipids, excess salts and reducing agents, and insoluble material, to name a few. The mistakes that are commonly made during sample preparation also include over-heating samples (temperature and time), using incorrect reducing agents, incorrect protein-to-sample buffer ratio, insoluble material in the sample, and over- and under-loading of protein sample.

Similarly, choosing the right gel chemistry for a specific research application can help minimize the validation time for that particular application. For example, neutral pH Bis-Tris gels are recommended for samples with low abundance of target proteins or when downstream applications require high protein integrity (mass spectrometry, posttranslational modification, or protein sequencing). Additionally, high molecular weight proteins (up to 500 kDa) can be optimally resolved by using Tris-acetate gels, whereas low molecular weight proteins (as low as 2.5 kDa) can be optimally resolved using Tricine gels. In this webinar, we will provide an overview of your options and guidance on how to get optimal protein resolution.

Learning objectives

  • Importance of sample preparation in SDS-PAGE electrophoresis
  • Importance of choosing the right gel chemistry for specific research applications

Webinar

Light up your western blots – fluorescent western blotting tips, tricks & more

paul-haney

Speaker:

Paul Haney, Ph.D.,
Senior Product Manager, Protein and Cell Analysis, Thermo Fisher Scientific

Biography:

Over the past 18 years, Paul Haney has been a key member of the R&D and product management team in the protein biology business unit of Thermo Fisher Scientific, where he has provided leadership and strategy for the development of products for the life science research market. He is focused on products for western blot detection, western blot and gel imaging, protein sample preparation, protein isolation, immunodetection, and mass spectrometry analysis. Before joining Thermo Fisher Scientific, Paul conducted postdoctorial research in receptor biology at the Mayo Clinic and in bioinformatics and protein stability at the University of Illinois/Urbana. Paul completed his PhD in molecular biology and protein biology at the University of Illinois/Urbana.

Abstract

While chemiluminescence has been the traditional approach to western blot detection, this strategy is being progressively supplemented or replaced with fluorescence-based detection. With the introduction of advanced CCD-camera–based imaging systems and scanning instruments, along with improvements in fluorescent conjugate technologies, scientists now have the necessary tools available to take advantage of the range of fluorescent dyes and antibodies for fluorescent western blot detection. Learn more about the best practices to help enable you to become successful at fluorescent western blotting.

Learning objectives

  • Understand the fundamentals of fluorescent western blotting
  • Successfully design fluorescent western blotting experiments

Poster presentation

Alternative blotting methods

Boguslawa “Bea” Dworecki, MS

Speaker:

Boguslawa “Bea” Dworecki, MS
Research Scientist, Protein and Cell Analysis, Thermo Fisher Scientific

Biography:

Bea is an R&D scientist in the protein analysis group. She completed her BS at Rockford University in 1997 in chemistry. She obtained her MS in medical biotechnology from the University of Illinois College of Medicine at Rockford in 2010. Over the past 20 years, she has worked on the development of numerous products for the life science research market. She has extensive experience in electrophoresis, protein transfer and detection, western blot and gel imaging, protein sample preparation, fluorescent labeling and purification of proteins and antibodies, dialysis, and protein staining and transfer devices.

Abstract

Western blotting is an important technique for characterizing specific protein targets. Successful western detection is dependent on the quality of protein transfer from polyacrylamide gels to the blotting membranes, and on the processing steps and reagents used. The traditional wet transfer method offers high transfer efficiency, but at the cost of time and effort. Improvements to dry and semi-dry transfer systems have greatly improved the transfer speed (less than 10 min) and convenience without compromising transfer efficiency. This poster will discuss the traditional wet blotting transfer technique as compared to dry and semi-dry transfer techniques.

Learning objectives

  • Compare the classical wet western blotting technique to dry and semi-dry blotting techniques
  • Learn about dry and semi-dry western blotting for high-efficiency transfer and quality results

Webinar

Anchored Protein Kinase-A (PKA) as a gatekeeper for the Connexin-43 gap junction

Guillaume Pidoux, PhD

Speaker:

Guillaume Pidoux, PhD
Senior Researcher at Inserm UMR-S 1180 (CRCN)

Biography:

Guillaume Pidoux completed his PhD at University Paris Descartes (Paris, France) in 2006 and did a postdoctoral fellowship at the Nordic EMBL, NCMM—University of Oslo (Oslo, Norway) in 2010. In 2012, he was recruited as a senior researcher at Inserm (French National Institute for Health and Medical Research). He is interested in the spatiotemporal regulation of cAMP signaling in physiological and pathophysiological conditions. He has recently established a small group at Inserm UMR-S 1180, which focuses on understanding the direct role of A-Kinase Anchoring Proteins (AKAPs) and Protein Kinase-A (PKA) in cardiac physiology and during the development of cardiomyopathies. With his cell biology and biochemistry approaches, he is paving the way to develop new therapeutic schemes aimed at improving the quality of life for patients.

Abstract

Anchored Protein Kinase-A (PKA) bound to A-Kinase Anchoring Protein (AKAP) mediates the effects of localized increases in cAMP in defined subcellular microdomains and retains the specificity in cAMP–PKA signaling to distinct extracellular stimuli. Gap junctions are pores between adjacent cells constituted by connexin (Cxs) proteins that provide a means of communication, electrical impulse propagation, and transfer of small molecules. One of the most widely and highly expressed Cxs is Cx43, which was shown to be regulated through phosphorylation by several kinases including PKA. Ezrin is a membrane-associated protein that can serve as an AKAP and hold an anchored pool of PKA. We found PKA to be part of a macromolecular signaling complex with ezrin and gap junction protein Cx43 that provides cAMP-mediated control of gap junction communication. We established that an inhibition of PKA activity or disruption of the ezrin–Cx43 interaction abolished PKA-dependent phosphorylation of Cx43 as well as gap junction communication. In vitro studies using peptide arrays, together with mass spectrometry, pointed to serine 369 and 373 of Cx43 as the major PKA phosphorylation sites. A combination of knockdown and reconstitution experiments and gap-FLIP assays with mutant Cx43 containing single or double phosphoserine–mimicking amino acid substitutions in putative PKA phosphorylation sites demonstrated that phosphorylation of S369 and S373 mediated gap junction communication. We propose that the PKA–ezrin–Cx43 macromolecular complex regulating gap junction communication constitutes a general mechanism to control opening of Cx43 gap junctions by phosphorylation in response to cAMP signaling in various cell types (i.e., heart, liver, and placenta).


Webinar

Complete and innovative western workflow solutions: filling the gap between art and science (presented in Chinese)

Jiao Yang, PhD

Speaker:

Jiao Yang, PhD
Technical Application Specialist at Thermo Fisher Scientific

Biography:

Jiao Yang works as a technical application specialist at Thermo Fisher Scientific in Shanghai, China. She received her PhD in biochemistry and molecular biology from Shanghai Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences. She has been working on the mechanism of protein degradation for about six years and has published a paper in cell research as a first-time author. In 2016, she joined Thermo Fisher Scientific and supports products in protein biology.

Abstract

Western blotting is a widely used and accepted technique to detect levels of protein expression in a cell or tissue extract. This webinar will cover the electrophoresis, transfer, and detection steps of the western blotting workflow, highlight our popular products, and introduce tips and tricks for superior western analysis to make western blotting more efficient and effective.

Learning objectives

  • Overview of the critical steps in protocols for western blot, electrophoresis, protein transfer, and western detection
  • Learn about our total western blotting solutions
  • Tips and tricks for superior western blot analysis

Poster presentation

Rapid Biuret based colorimetric assay for protein quantitation

Ramesh Ganapathy, PhD

Speaker:

Ramesh Ganapathy, PhD
R&D Scientist III, Protein and Cell Analysis, Thermo Fisher Scientific

Biography:

Ramesh Ganapathy joined Thermo Fisher Scientific in 2001 after completing his PhD at the University of Wisconsin-Madison and a postdoctoral fellowship at the Department of Bioengineering at the University of Washington-Seattle. He also holds his MBA degree from Northern Illinois University. Since joining Thermo Fisher Scientific, he has worked on developing a broad range of products including resins, substrates for western blotting, magnetic beads, and peptide and protein assay products.

Abstract

Protein quantitation assays are essential for workflows that include protein extraction, labeling, and analysis. The simplest method for protein quantitation is measuring the absorbance of a protein sample at 280 nm (A280) and calculating the quantity using the Beer Lambert Law. However, this method is only applicable when the sample is a purified protein solution. Most cell biology experiments result in mixed or unpurified protein samples extracted from cell or tissue lysates. The Bradford assay (or Coomassie dye–based assay) has been a standard protein quantitation assay for unpurified proteins for some time, but a major drawback of this assay is its poor linearity. An improved assay, the Thermo Scientific Pierce BCA Protein Assay using bicinchoninic acid, is a chelator-based assay with exceptional linearity and low protein-to-protein variability that was quickly established as the “gold standard” for protein assays. One drawback with this assay, though, is the relatively long incubation time (30 minutes at 37˚C or 2 hours at room temperature). This poster presents the development of an improved BCA protein quantitation assay, the new Thermo Scientific Pierce Rapid Gold BCA Protein Assay, that addresses the need for a shorter incubation time (5 minutes at room temperature). Herein we present our evaluation of protein-to-protein variability, accuracy of “known” protein mixes, and closeness in protein estimation for this new assay as compared to the classic BCA assay. The results of this evaluation demonstrate that the new Pierce Rapid Gold BCA assay with the shorter incubation time provides the same accuracy as the original BCA assay.

Learning objectives:

  • A rapid way to quantitate protein using the same principle as the traditional BCA assay

Webinar

Protein sample evaluation using the NanoDrop One UV-Vis Spectrophotometer

Voula Kodoyianni, PhD

Speaker:

Voula Kodoyianni, PhD
Product Marketing Manager—NanoDrop instruments, Thermo Fisher ScientificThermo Fisher Scientific

Biography:

Voula Kodoyianni is the product manager for Thermo Scientific NanoDrop microvolume UV-Vis instruments and led the development and market launch of the NanoDrop One UV-Vis Spectrophotometer in 2015. Prior to joining Thermo Fisher Scientific, Dr. Kodoyianni was the chief scientific officer for GWC Technologies, led a genome sequencing team at the University of Wisconsin-Madison, and was a Howard Hughes Medical Institute Fellow conducting research in C. elegans development. She holds a master’s degree in chemistry from Duke University and a PhD in biochemistry from the University of Wisconsin-Madison.

Abstract

Protein sample evaluation is an important step in many protein workflows. The NanoDrop One microvolume spectrophotometer supports protein sample quantification with multiple preconfigured applications, including direct absorbance at A280, A205, or other wavelengths using the Thermo Scientific Pierce colorimetric assays (BCA, Rapid Gold BCA, Pierce 660, Bradford, etc.) and a proteins and labels application for quantitation of metalloproteins and fluorescently labeled proteins. We will review what you need to consider when choosing a protein quantification method for your sample. Is the sample one purified protein or a mixture of proteins? Does your peptide contain tyrosine and tryptophan residues? Does the sample buffer absorb in the UV spectrum? How can co-purified DNA affect your protein sample measurement? What is a good blank/control for your sample? Answers to these questions and other key considerations will be discussed.

Learning objectives:

  • Understand protein extinction coefficients—how to obtain the most accurate protein sample quantification using UV absorbance at 280 nm
  • Learn how new Thermo Scientific Acclaro sample intelligence technology in the NanoDrop One spectrophotometer delivers information not only about the concentration but also the quality of your protein sample

Poster presentation

Development of Multiplex xMAP Technology-Based Assays for Simultaneous Detection of Soluble Checkpoint Molecules Involved in Anti-Cancer Immune Response

Susanne Oehler, PhD

Speaker:

Susanne Oehler, PhD
Senior Manager, Research and Development, Antibodies and Immunoassays, Thermo Fisher Scientific

Biography:

Susanne received her PhD at the University of Vienna where she characterized the gene of a major cytoskeletal interlinking protein, plectin, which affects dynamic properties of the cytoskeleton and plays a role in tumor biology. Her postgraduate work focusing on chemotherapy resistance mechanisms in breast cancer cells was carried out at the Ludwig Bolzmann Institute of Clinical Experimental Oncology at the General Hospital in Vienna. Currently Suzanne is a senior scientist in the antibodies and immunoassays group in R&D at Thermo Fisher Scientific. She is a group leader in the multiplex immunoassays development team.

Abstract

To ensure the immune system’s balance between the recognition of non-self and the prevention of autoimmunity, the activity of immune cells needs to be strictly controlled. During the process of carcinogenesis, tumor cells try to resist immune responses and work to suppress the cycle of cancer immunity through multiple mechanisms involving regulatory proteins. These proteins—referred to as immune checkpoints—can affect immunoregulatory pathways by either boosting (co-stimulatory) or restricting (co-inhibitory) the immune response. For the majority of these checkpoint markers, soluble isoforms or shed variants that function as immune adjuvants or decoy receptors, have been identified. To date, cancer therapies that use antibodies targeted to the proteins within these immune checkpoint pathways have proven effective. It is also hypothesized that the measurement of the circulating concentration of the soluble forms of the immune checkpoint proteins could potentially correlate with the clinical efficacy of corresponding checkpoint modulator drugs.

To test this hypothesis, we developed two comprehensive multiplexing panels (using Luminex® xMAP® technology) that are specifically designed to detect soluble checkpoint molecules involved in T cell and NK cell regulation. The panels allow the simultaneous detection of immune stimulatory and inhibitory factors, allowing a more holistic picture of the molecular players in cancer immunity. Preliminary results using the Invitrogen Immuno-Oncology Checkpoint 14-Plex Human ProcartaPlex Panel 1 (Cat. No. EPX14A-15803-901) and the Invitrogen Immuno-Oncology Checkpoint 14-Plex Human ProcartaPlex Panel 2 (Cat. No. EPX140-15815-901) demonstrate that there are different expression patterns for each that are dependent on both disease state and patient treatment. More in-depth analysis of these soluble biomarkers will hopefully shed more light on the biology of the immune checkpoint pathways and provide a possible tool for monitoring response to therapeutic treatment.

Learning objectives:

  • Investigate the advantages of using soluble checkpoint molecules as potential markers for the response to chemotherapy
  • Develop immunoassays for new immune regulating targets

Webinar

Invitrogen immunoassays: vast choice of appropriate tools for specific needs

Markus Miholits, MS

Speaker:

Markus Miholits, MS
Senior Manager, Research and Development, Antibodies and Immunoassays, Thermo Fisher Scientific

Biography:

Markus leads the R&D group at the Vienna Center of Excellence for Immunoassay Development at Thermo Fisher Scientific. He is responsible for product and technology development as well as contract and custom lab services. Prior to joining Thermo Fisher Scientific, Markus was head of the laboratory for immunoassay development at Affymetrix and was group leader for immunoassay development at eBioscience. Also, he was project leader of R&D for immunoassay development and establishment of new immunoassay technologies (instant ELISA assay; in-house developed, bead-based multiplexing immunoassay technology) at Bender MedSystems GmbH. Markus holds a master’s degree from the University of Vienna (Institute of Biochemistry).

Abstract

Understanding the complex, yet coordinated protein interactions that sustain life, may help unlock the mysteries of disease mechanisms and thereby treatments. Protein identification and quantification have become extremely valuable in advancing our understanding and finding cures. As there are many methodologies available to scientists today, it can be challenging to choose which tools are the most effective to answer specific questions.

Based on the decades of market experience in the immunological research reagents market, Thermo Fisher Scientific addresses different customer needs by different technologies for detecting the same analytes and offers a vast choice of appropriate tools for the customer’s specific needs.

Learning objectives:

  • Identify time-saving technologies and tools for protein quantitation
  • Understand the benefits of choosing from different immunoassay platforms and formats, all from the same provider

Webinar

ProQuantum High-Sensitivity Immunoassays offer minimum sample consumption With maximum performance

David Bourdon, PhD

Speaker:

David Bourdon, PhD
Senior R&D Manager, Antibodies and Immunoassays, Thermo Fisher Scientific

Biography:

David’s PhD and postdoctoral work focused on the study of G protein–coupled receptor signaling and thrombosis. Following his academic training, he led multiplexed immunoassay automation efforts at a Luminex partnering company. Joining Thermo Fisher Scientific in 2009, David is now a senior R&D manager focusing on next-generation immunoassay development including the Invitrogen ProQuantum platform. David and his team continue working with translational investigators to identify serum-based biomarkers in cancer, autoimmunity, and inflammation.

Abstract

Early detection of protein biomarkers is critical to the study of human diseases, yet current tools available for low-level protein quantitation do not address all the needs for protein and molecular biologists alike. A common concern is sample volume limitation due to difficulties in obtaining sufficient biological samples from various models in research studies. Additionally, cost and time-to-answer are always a factor when considering which assay platform to use. In many cases, end users must make trade-offs on which factors impact their work the least.

Invitrogen ProQuantum High-Sensitivity Immunoassays combine multiple benefits into a single platform so that users don’t need to make trade-offs. The platform works by combining the analyte specificity of high-affinity antibody–antigen binding with the signal detection and amplification of real-time PCR. The result is an assay that can measure low-expressing proteins (femtogram levels) while only consuming a fraction of sample volume (i.e.,1–2 microliters per sample) compared to other immunoassay methods. Best of all, it uses a streamlined workflow (2 hours) that is highly scalable and affordable on an open, widely used qPCR platform.

Learning objectives:

  • To understand the multiple benefits of ProQuantum High-Sensitivity Immunoassays and how the platform works
  • To understand how the kits are designed for a simplified workflow amenable to high-throughput applications

Webinar

Signaling pathways at your fingertips – multiplex IP to targeted mass spectrometry assay kits for AKT/mTOR pathway

Bhavin Patel, MD, MS

Speaker:

Bhavin Patel, MD, MS
Senior R&D Scientist, Protein and Cell Analysis, Thermo Fisher Scientific

Biography:

Bhavin Patel works as a senior R&D scientist in mass spectrometry reagents group at Thermo Fisher Scientific. Bhavin has an MD degree from the Maharaja Sayajirao University of Baroda in India, and an MS in biotechnology from the Georgetown University. Bhavin had led a protein biomarker assay group at NextGen Sciences and a proteomics/mass spectrometry group at Fox Chase Cancer Center before joining Thermo Fisher Scientific in 2013. Since joining Thermo Fisher Scientific, Bhavin has led the development of targeted mass spectrometry (MS) assay kits, new MS standards, protein sample preparation reagents, an antibody validation method for IP-MS, and reagents for quantitative proteomic analysis.

Abstract

Proteomics offers a deeper understanding of the protein events influencing cellular and biological function. However, identifying key regulated proteins and their implicated signaling pathways represents a major analytical challenge to researchers. This is due to the broad dynamic range of protein abundance in biological samples and the inherent limitations of the instrumentation and methodologies utilized to probe and detect minute target proteins in complex cellular environments. In this webinar, we discuss targeted mass spectrometry (MS)-based approaches and the various strategies designed to reduce sample complexity through improved sample preparation and enrichment. Focusing on proteins involved in the AKT/mTOR signaling pathway, we provide guidance toward targeted MS assay development and validation, including robustness, precision, and accuracy of these assays. Finally, we’ll examine results of multiplexed IP to targeted MS assays compared to existing immunoassay techniques.

Learning objectives:

  • Development and validation of immunoenrichment to mass spectrometry (IP-MS) assays
  • Accurate and precise quantitation of multiple AKT/mTOR pathway proteins by multiplex IP to targeted MS assays and comparison to exiting immunoassay techniques

Webinar

Addressing the challenges of bioconjugation for improved crosslinking and modification

Greg Hermanson

Speaker:

Greg Hermanson
Chief Technology Officer and Principal at Aurora Microarray Solutions, Inc. & President of Greg T. Hermanson, Inc.

Biography:

Greg Hermanson is the chief technology officer and principal at Aurora Microarray Solutions, Inc. and the president of Greg T. Hermanson, Inc., a bioscience consulting company. Greg has over three decades of experience in the development of life science research and diagnostic products, with particular expertise in protein chemistry, assay development, immobilization, and bioconjugation. He is well known as the author of Bioconjugate Techniques, (now in its third edition), which is an extensive manual on the methods of bioconjugation. Greg has had a significant impact on the bioscience fields as proven by nearly 27,500 citations of his publications and patents.

Abstract

Bioconjugation is a critical technique for creating many of the key reagents used in research and commercial applications. This webinar will cover in great detail the major reactions used to form bioconjugates as well as the functional groups which react with them. This includes identifying the principle target groups on proteins, oligonucleotides, glycans, and carbohydrates and matching them with the appropriate reactive groups for conjugation. The reactive groups covered will include those useful for amine conjugations, thiol reactions, carbonyl reactions, and for bioorthogonal coupling.

This webinar will examine the major products of these reactions, as well as any potential side reactions or competing hydrolysis that may occur, and any interfering substances that should be avoided. The goal of this webinar is to provide a solid understanding of these reactions and how to optimize them to create the best possible bioconjugates for the intended downstream applications.

Learning objectives

  • Learn about the major reactive groups used in bioconjugation and how to optimize each reaction
  • Learn to identify major products of each reaction and understand the effects of hydrolysis, side reactions, and interfering substances
  • Discuss tips and tricks for improved results