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Poster presentation

In vitro research method for screening inhibitors of protein translation

Speaker:

Krishna Vattem, Ph.D.
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

In vitro translation (IVT), or cell-free expression, offers a unique and powerful research tool to screen for translational inhibitors that regulate both cellular and viral protein expression. Here we discuss a simple, quick method to identify inhibitors of both cap-dependent and cap-independent protein translation, using the Thermo Scientific 1-Step Human Coupled IVT Kit. Rapid assay readout (60–90 min), amenability for miniaturization, and insensitivity to compound toxicity make in vitro translation an attractive alternative to cell-based screens for high-throughput screening (HTS) of novel inhibitors of protein synthesis.

Learning Objectives

  • Understand how In vitro translation systems offer a novel approach to studying protein synthesis inhibition.
  • Demonstrate how the 1-Step Human Coupled IVT Kit can be utilized for high-throughput screening of protein synthesis inhibitors in a rapid, miniaturized format.
  • Identify the unique advantages of using dual luciferase assays

Poster presentation

High capacity magnetic supports for automated antibody and epitope-tagged protein purifications

Speaker:

Barbara Kaboord, Ph.D.
Senior R & D Manager, Protein and Cell Analysis, Thermo Fisher Scientific

Biography:

Barbara received her Ph.D. in biochemistry from the Medical College of Wisconsin. Her post-doctoral fellowship was performed at The Pennsylvania State University under the direction of Stephen Benkovic. She has over twenty years of industry experience, and she currently leads a team of R&D scientists in new product development in the areas of protein interactions, affinity chromatography, and protein sample preparation.

Abstract

For academic, biotech, and pharmaceutical scientists who are screening clones or performing high throughput protein purification, the goal is to automate the sample processing without sacrificing binding capacity. Traditionally, magnetic beads have facilitated process automation, but they lack the high binding capacity of agarose resins. On the other hand, agarose or sepharose resins have high binding capacity, but are not amenable to automation. Magnetic agarose resins combine the best of both formats by enabling high throughput sample processing with high protein binding capacity. Here we demonstrate the advantages of using magnetic agarose resins in simple benchtop  protein purifications as well as their utility in 1) the screening of recombinant antibodies using the Invitrogen ExpiCHO Expression Systems and 2) the purification of recombinant proteins expressed by in vitro translation (cell-free) systems using the Thermo Scientific KingFisher Flex magnetic particle processor. Both the Protein AG and anti-DYKDDDDK magnetic agarose supports result in isolation of >0.5mg protein per sample at >90% purity.

Learning Objectives

  • Become familiar with the use of cell based and cell free systems for protein expression.
  • Determine how to use magnetic affinity supports to screen best expressers.
  • Learn how to automate sample processing with magnetic beads for protein purification

Poster presentation

Antibody purification: Development of two new highly efficient purification resins

Speaker:

Aaron McBride,
R&D Scientist III, Protein and Cell Analysis, Thermo Fisher Scientific

Biography:

Aaron McBride received his MS in cell and molecular biology from Michigan State University. He has played an instrumental role in the development, commercialization and support of multiple chromatography resins and recombinant proteins introduced over the past seven years at Thermo Fisher Scientific. His development work also includes products for protein purification, molecular biology and in vitro translation.

Abstract

Academic, biotech, and pharmaceutical scientists all have different requirements for their purified antibodies based on their final application. While these requirements will ultimately be satisfied by additional purification steps, the capture step is relatively the same. Most antibodies will be captured by one of four different affinity ligands, Protein A, G, A/G or L, with Protein A being the most utilized. The decision of which ligand to use is typically based on antibody species, binding affinity for the ligand, relative stability of the antibody that is being purified, and chemical stability. The ligand is only one half of the equation of which resin to choose. The other half is the chromatography support on which the ligand is immobilized. The chromatography support can influence final purity, maximum flow rates, elution volume, and binding capacity at different flow rates, all of which impact the total efficiency of the purified protein recovery. In this poster we describe the development of Protein G and Protein A/G on Applied Biosystems POROS perfusion chromatography media. Both resins provide up to four fold higher binding capacity at higher flow rates when compared to similar agarose-based resins. Another unique advantage of POROS perfusion media is that it provides better mass transfer which results in smaller elution volume. In addition, both resins have low leaching and similar chemical compatibility to other Protein G and A/G agarose resins.


Poster presentation

Efficient and convenient enrichment of multi-spanning membrane proteins for proteomic studies

Speaker:

Joanna Geddes,
R&D Scientist II, Protein and Cell Analysis, Thermo Fisher Scientific

Biography:

Joanna received her BS in microbiology from Western Illinois University. She has actively worked on the development, commercialization and support of protein sample preparation and protein interaction products introduced over the past eight years. This also includes development of products for dialysis, protein concentration, membrane protein extraction, protease and phosphatase inhibition, and immunoprecipitation. She has experience in a multitude of techniques such as mass spectrometry sample prep, cell and tissue lysis, cell separation, and protein expression and extraction.

Abstract

Examining the membrane proteome is vital to understand its role in normal and disease function. However, the isolation and extraction of multi-spanning membrane proteins for proteomic study often proves difficult. Traditional isolation methods are tedious and time-consuming. Additional drawbacks include poor solubilization, incompatibility with downstream applications, and disruption of membrane protein complexes. In this study, we report a procedure that enables fast, convenient solubilization of membrane proteins from cultured cells and tissue. This sequential detergent extraction method allows for the enrichment of both integral membrane proteins and membrane-associated proteins. To validate our method, three integral multi-spanning membrane proteins (ADP/ATP translocase 3 (SLC25A6), sodium/potassium ATPase alpha subunit (AT1A1) and adenylate cyclase 2 (ADCY2)) were evaluated. Membrane protein extraction in cell lines (A431, HeLa, HCT116, HepG2, HEK293 and A549) and mouse tissue (brain, kidney and liver) using this reagent-based extraction outperforms the other available kits as determined by western blotting and mass spectrometry. Additionally, extraction of multi-spanning membrane protein is improved by increasing the ionic strength of the solubilization buffer, obtaining 70-85% extraction, as confirmed by western blot densitometry. Native membrane protein complexes are also preserved, allowing for compatibility with co-immunoprecipitation experiments. This straight forward and robust method enables researchers to better investigate the role of membrane proteins in cellular functions.

Webinar

Choosing the right protein gel for your research application

Speaker:

Alok Tomar, Ph.D.
Product Manager, Protein and Cell Analysis, Thermo Fisher Scientific

Biography:

Alok’s Ph.D. 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 5 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 Thermo Fisher Scientific protein gel electrophoresis portfolio and has recently launched the Invitrogen Novex Tris-Glycine Plus Midi gels that provide reproducible quality and performance.

Abstract

It is widely perceived that protein pre-cast or hand-cast gels are chemically inert and do not cause protein modifications during electrophoresis.  However, multiple publications have reported chemical modification of amino acid side chains during separation of proteins by electrophoresis, including deamidation, aspartate–proline bond cleavage, methionine and tryptophan oxidation, and the Michael addition of sulfhydryl or amino groups to the double bond of acrylamide that is not polymerized into the gel matrix. These modifications can change the electrophoretic mobility of proteins resulting in blurred or multiple bands and are detrimental for downstream research applications. For example, mass spectrometry of the modified protein sample results in the predicted peptides appearing at several masses due to the diversity of unwanted modifications.

Choosing the right gel chemistry for a specific research application can help minimize protein modification. 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). Similarly, 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. This webinar will highlight the different protein modifications occurring during electrophoresis, the importance of pH in sample integrity, how to choose the right gel chemistry for specific research applications and how to introduce the right new gel chemistry in your research workflow.

Learning objectives

  • How to choose the right pre-cast gel chemistry for your specific research application.
  • Why Bis-Tris gel chemistry is ideal for post-translational modification applications.
  • Why Tris-glycine gel chemistry is not ideal for all research applications.

Webinar

Classical and modernized western blotting

Speaker:

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

Biography:

Over the past 17 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. Development 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 post doctorial research in receptor biology at the Mayo Clinic and in bioinformatics and protein stability at the University of Illinois/Urbana. Paul completed his Ph.D. in molecular biology and protein biology at the University of Illinois/Urbana.

Abstract

Western blotting is a fundamental research application. Although simple in nature, western blotting can be considered an artwork—researchers have put their own tweaks on the approach in order to get the best possible results. This webinar will cover the electrophoresis, transfer, and detection steps of the western blotting workflow, in the framework of the classical approach and the modernized twists that make western blotting more efficient and effective. 

Learning objectives

  • Review the classical western blotting technique as a foundational research tool
  • Learn about modern twists on classical western blotting which improve efficiency and quality of results

Webinar

Chemiluminescent western blot detection: Bright and bold detection no matter how scarce your target

Speaker:

Emily Halbrader
Product Manager, Protein and Cell Analysis, Thermo Fisher Scientific

Biography:

Emily is the product manager for protein detection reagents and systems in Biosciences Division at Thermo Fisher Scientific. She has been with the company for 2 years, coming from a product management career in the health care industry and an academic background in biological sciences. She currently manages a product portfolio for protein detection including western blotting reagents and kits, protein quantitation assays, and assay development reagents. In addition, Emily dedicates much of her time to interacting with customers across the academic, industrial and diagnostic markets and working with the Research and Development teams on new product development.

Abstract

Chemiluminescent detection is a widely-used method for detecting target proteins in a western blotting. This detection method is the result of an enzyme reaction of horseradish peroxidase (HRP) or alkaline phosphatase (AP) which produces light, and can be measured by use of x-ray film or a CCD imager. Chemiluminescent substrates allow for various advantages over other methods in detection including, but not limited to, increased sensitivity, high signal-to noise ratios (or low background), and the ability to strip and reprobe the blot. Contrary to popular belief, however, "one size" does not fit all" when it comes to western blotting substrates. With so many options on the market, it is sometimes difficult to determine which substrate performs the best for individual western blot systems. In this webinar we will discuss the best methods for optimizing this technique as well as how to determine the appropriate reagents for your sensitive target detection.

Webinar

Protein concentration assays – novel, simplified and rapid techniques for quantifying proteins in solution

Speaker:

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

Biography:

Ramesh Ganapathy joined Thermo Fisher Scientific in 2001 after completing his Ph.D. at University of Wisconsin-Madison and a post-doctoral fellowship at the Department of Bioengineering at 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 ranging from resins, substrates for western blotting, magnetic beads, and peptide and protein assay products.

Abstract

Protein concentration assays are a widely used technique in protein research to determine the total amount of protein in a sample prior to downstream applications.  While there are many protein assay options available for scientists, it is important to choose one that delivers the most accurate results based on the sample type, protein preparation methodologies and downstream applications. New developments and a recently introduced product in this area now provide scientists the full combination of quality and ease-of-use by offering high accuracy and reproducibility. In this webinar, we will discuss the principles behind the most popular protein concentration assays, how to achieve the highest quality results with your sample, and the importance of using the proper standards, protein preparation, sample composition, and downstream applications in choosing the right protein assay for your needs.

Learning objectives:

  • Choose the best colorimetric protein assay based on sample composition.
  • Understand the differences between dye-based (Bradford, Pierce 660 nm assay) and Copper-chelating (BCA) assays.
  • Describe the different instruments used with protein assays

Webinar

Harnessing the power of TMT11plex sample multiplexing and improved phosphopeptide enrichment to gain new insights into signaling pathways

Speaker:

Ryan Bomgarden, Ph.D.
Senior R&D Staff Scientist, Protein and Cell Analysis, Thermo Fisher Scientific

Biography:

Ryan Bomgarden received his bachelor’s degree from Coe College with majors in chemistry and molecular biology. He received his Ph.D. at Stanford University under Dr. Karlene Cimprich where he worked on biochemical characterization of the checkpoint kinase ATR DNA binding activity and signaling in UV-sensitive human cell lines. Since joining Thermo Fisher Scientific in protein biology R&D, he has worked on the development of key protein research reagents including photo-reactive crosslinkers, SILAC metabolic labeling kits and subcellular protein fractionation kits. Currently Dr. Bomgarden is in the mass spectrometry R&D group where his development work includes tandem Mass Tag reagents for relative quantitation of proteomic samples, HeLa protein digest standards for LC-MS QC, MS-cleaveable crosslinkers for protein structure analysis, heavy protein IVT kits for production of stable isotope-labeled proteins; and Thermo Scientific ActivX probes for kinase, GTPase and serine hydrolase inhibitor profiling and detection. In addition to his work at Thermo Fisher Scientific, Dr. Bomgarden is also an adjunct professor at the University of Illinois College of Medicine where he teaches courses on biochemical techniques, drug design and ethics. Dr. Bomgarden is also heavily involved in STEM education, creating and performing science demonstrations for local schools and non-profit organizations.

Abstract

Mass spectrometry (MS) has become the predominant technology to analyze proteins due to its ability to identify and characterize proteins and their modifications with high sensitivity and selectivity.  Increasing the number of samples analyzed simultaneously in a single mass spectrometry experiment is essential for increased sample throughput, fewer missing measurements between samples, and increased statistical power among replicates.  Thermo Scientific Tandem Mass Tag TMT Reagents are one technology which enables concurrent identification and multiplexed quantitation of different samples using tandem mass spectrometry. Recently, we have extended the multiplexing capabilities of TMT Reagents from 6 to 11 without an increase in the tag size or structure by utilizing the mass difference between 13C and 15N isotopes in the reporter region.  We have also developed new phosphopeptide enrichment workflows with improved phosphopeptide yield, selectivity and identification rates.  Overall, these additions to the TMT workflow enable higher sample multiplexing and provide in depth quantification of protein post-translational modifications.

Learning objectives:

  • Understand the basics of how tandem mass tags can be used for protein multiplexing 
  • Discuss how the multiplexing capabilities of TMT Reagents were extended from 6 to 11 without an increase in the tag size or structure 
  • Identify the differences between Fe-NTA and TiO2 and how to use sequentially for maximum phosphopeptide enrichment

Webinar

Strategies for successful crosslinking and bioconjugation applications

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 significant impact on the bioscience fields as proven by nearly 27,500 citations to his publications and patents.

Abstract

Crosslinking and bioconjugation reagents are widely used in research to study protein interactions, or as tools for assay or nanoparticle development. While there are many reactive chemistries and labeling tags available to scientists, it is important to choose the tools that deliver the desired outcome based on the functional groups available, steric considerations, cleavability and solubility required for successful conjugation. In this webinar, we will discuss the basics of crosslinking, and simple guidelines on how to achieve the most efficient modification for some typical applications, including labeling antibodies, immobilizing biomolecules to surfaces, and capturing protein interactions for mass spectrometry analysis.

Poster presentation

In vitro research method for screening inhibitors of protein translation

Speaker:

Krishna Vattem, Ph.D.
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

In vitro translation (IVT), or cell-free expression, offers a unique and powerful research tool to screen for translational inhibitors that regulate both cellular and viral protein expression. Here we discuss a simple, quick method to identify inhibitors of both cap-dependent and cap-independent protein translation, using the Thermo Scientific 1-Step Human Coupled IVT Kit. Rapid assay readout (60–90 min), amenability for miniaturization, and insensitivity to compound toxicity make in vitro translation an attractive alternative to cell-based screens for high-throughput screening (HTS) of novel inhibitors of protein synthesis.

Learning Objectives

  • Understand how In vitro translation systems offer a novel approach to studying protein synthesis inhibition.
  • Demonstrate how the 1-Step Human Coupled IVT Kit can be utilized for high-throughput screening of protein synthesis inhibitors in a rapid, miniaturized format.
  • Identify the unique advantages of using dual luciferase assays

Poster presentation

High capacity magnetic supports for automated antibody and epitope-tagged protein purifications

Speaker:

Barbara Kaboord, Ph.D.
Senior R & D Manager, Protein and Cell Analysis, Thermo Fisher Scientific

Biography:

Barbara received her Ph.D. in biochemistry from the Medical College of Wisconsin. Her post-doctoral fellowship was performed at The Pennsylvania State University under the direction of Stephen Benkovic. She has over twenty years of industry experience, and she currently leads a team of R&D scientists in new product development in the areas of protein interactions, affinity chromatography, and protein sample preparation.

Abstract

For academic, biotech, and pharmaceutical scientists who are screening clones or performing high throughput protein purification, the goal is to automate the sample processing without sacrificing binding capacity. Traditionally, magnetic beads have facilitated process automation, but they lack the high binding capacity of agarose resins. On the other hand, agarose or sepharose resins have high binding capacity, but are not amenable to automation. Magnetic agarose resins combine the best of both formats by enabling high throughput sample processing with high protein binding capacity. Here we demonstrate the advantages of using magnetic agarose resins in simple benchtop  protein purifications as well as their utility in 1) the screening of recombinant antibodies using the Invitrogen ExpiCHO Expression Systems and 2) the purification of recombinant proteins expressed by in vitro translation (cell-free) systems using the Thermo Scientific KingFisher Flex magnetic particle processor. Both the Protein AG and anti-DYKDDDDK magnetic agarose supports result in isolation of >0.5mg protein per sample at >90% purity.

Learning Objectives

  • Become familiar with the use of cell based and cell free systems for protein expression.
  • Determine how to use magnetic affinity supports to screen best expressers.
  • Learn how to automate sample processing with magnetic beads for protein purification

Poster presentation

Antibody purification: Development of two new highly efficient purification resins

Speaker:

Aaron McBride,
R&D Scientist III, Protein and Cell Analysis, Thermo Fisher Scientific

Biography:

Aaron McBride received his MS in cell and molecular biology from Michigan State University. He has played an instrumental role in the development, commercialization and support of multiple chromatography resins and recombinant proteins introduced over the past seven years at Thermo Fisher Scientific. His development work also includes products for protein purification, molecular biology and in vitro translation.

Abstract

Academic, biotech, and pharmaceutical scientists all have different requirements for their purified antibodies based on their final application. While these requirements will ultimately be satisfied by additional purification steps, the capture step is relatively the same. Most antibodies will be captured by one of four different affinity ligands, Protein A, G, A/G or L, with Protein A being the most utilized. The decision of which ligand to use is typically based on antibody species, binding affinity for the ligand, relative stability of the antibody that is being purified, and chemical stability. The ligand is only one half of the equation of which resin to choose. The other half is the chromatography support on which the ligand is immobilized. The chromatography support can influence final purity, maximum flow rates, elution volume, and binding capacity at different flow rates, all of which impact the total efficiency of the purified protein recovery. In this poster we describe the development of Protein G and Protein A/G on Applied Biosystems POROS perfusion chromatography media. Both resins provide up to four fold higher binding capacity at higher flow rates when compared to similar agarose-based resins. Another unique advantage of POROS perfusion media is that it provides better mass transfer which results in smaller elution volume. In addition, both resins have low leaching and similar chemical compatibility to other Protein G and A/G agarose resins.


Poster presentation

Efficient and convenient enrichment of multi-spanning membrane proteins for proteomic studies

Speaker:

Joanna Geddes,
R&D Scientist II, Protein and Cell Analysis, Thermo Fisher Scientific

Biography:

Joanna received her BS in microbiology from Western Illinois University. She has actively worked on the development, commercialization and support of protein sample preparation and protein interaction products introduced over the past eight years. This also includes development of products for dialysis, protein concentration, membrane protein extraction, protease and phosphatase inhibition, and immunoprecipitation. She has experience in a multitude of techniques such as mass spectrometry sample prep, cell and tissue lysis, cell separation, and protein expression and extraction.

Abstract

Examining the membrane proteome is vital to understand its role in normal and disease function. However, the isolation and extraction of multi-spanning membrane proteins for proteomic study often proves difficult. Traditional isolation methods are tedious and time-consuming. Additional drawbacks include poor solubilization, incompatibility with downstream applications, and disruption of membrane protein complexes. In this study, we report a procedure that enables fast, convenient solubilization of membrane proteins from cultured cells and tissue. This sequential detergent extraction method allows for the enrichment of both integral membrane proteins and membrane-associated proteins. To validate our method, three integral multi-spanning membrane proteins (ADP/ATP translocase 3 (SLC25A6), sodium/potassium ATPase alpha subunit (AT1A1) and adenylate cyclase 2 (ADCY2)) were evaluated. Membrane protein extraction in cell lines (A431, HeLa, HCT116, HepG2, HEK293 and A549) and mouse tissue (brain, kidney and liver) using this reagent-based extraction outperforms the other available kits as determined by western blotting and mass spectrometry. Additionally, extraction of multi-spanning membrane protein is improved by increasing the ionic strength of the solubilization buffer, obtaining 70-85% extraction, as confirmed by western blot densitometry. Native membrane protein complexes are also preserved, allowing for compatibility with co-immunoprecipitation experiments. This straight forward and robust method enables researchers to better investigate the role of membrane proteins in cellular functions.

Webinar

Choosing the right protein gel for your research application

Speaker:

Alok Tomar, Ph.D.
Product Manager, Protein and Cell Analysis, Thermo Fisher Scientific

Biography:

Alok’s Ph.D. 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 5 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 Thermo Fisher Scientific protein gel electrophoresis portfolio and has recently launched the Invitrogen Novex Tris-Glycine Plus Midi gels that provide reproducible quality and performance.

Abstract

It is widely perceived that protein pre-cast or hand-cast gels are chemically inert and do not cause protein modifications during electrophoresis.  However, multiple publications have reported chemical modification of amino acid side chains during separation of proteins by electrophoresis, including deamidation, aspartate–proline bond cleavage, methionine and tryptophan oxidation, and the Michael addition of sulfhydryl or amino groups to the double bond of acrylamide that is not polymerized into the gel matrix. These modifications can change the electrophoretic mobility of proteins resulting in blurred or multiple bands and are detrimental for downstream research applications. For example, mass spectrometry of the modified protein sample results in the predicted peptides appearing at several masses due to the diversity of unwanted modifications.

Choosing the right gel chemistry for a specific research application can help minimize protein modification. 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). Similarly, 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. This webinar will highlight the different protein modifications occurring during electrophoresis, the importance of pH in sample integrity, how to choose the right gel chemistry for specific research applications and how to introduce the right new gel chemistry in your research workflow.

Learning objectives

  • How to choose the right pre-cast gel chemistry for your specific research application.
  • Why Bis-Tris gel chemistry is ideal for post-translational modification applications.
  • Why Tris-glycine gel chemistry is not ideal for all research applications.

Webinar

Classical and modernized western blotting

Speaker:

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

Biography:

Over the past 17 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. Development 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 post doctorial research in receptor biology at the Mayo Clinic and in bioinformatics and protein stability at the University of Illinois/Urbana. Paul completed his Ph.D. in molecular biology and protein biology at the University of Illinois/Urbana.

Abstract

Western blotting is a fundamental research application. Although simple in nature, western blotting can be considered an artwork—researchers have put their own tweaks on the approach in order to get the best possible results. This webinar will cover the electrophoresis, transfer, and detection steps of the western blotting workflow, in the framework of the classical approach and the modernized twists that make western blotting more efficient and effective. 

Learning objectives

  • Review the classical western blotting technique as a foundational research tool
  • Learn about modern twists on classical western blotting which improve efficiency and quality of results

Webinar

Chemiluminescent western blot detection: Bright and bold detection no matter how scarce your target

Speaker:

Emily Halbrader
Product Manager, Protein and Cell Analysis, Thermo Fisher Scientific

Biography:

Emily is the product manager for protein detection reagents and systems in Biosciences Division at Thermo Fisher Scientific. She has been with the company for 2 years, coming from a product management career in the health care industry and an academic background in biological sciences. She currently manages a product portfolio for protein detection including western blotting reagents and kits, protein quantitation assays, and assay development reagents. In addition, Emily dedicates much of her time to interacting with customers across the academic, industrial and diagnostic markets and working with the Research and Development teams on new product development.

Abstract

Chemiluminescent detection is a widely-used method for detecting target proteins in a western blotting. This detection method is the result of an enzyme reaction of horseradish peroxidase (HRP) or alkaline phosphatase (AP) which produces light, and can be measured by use of x-ray film or a CCD imager. Chemiluminescent substrates allow for various advantages over other methods in detection including, but not limited to, increased sensitivity, high signal-to noise ratios (or low background), and the ability to strip and reprobe the blot. Contrary to popular belief, however, "one size" does not fit all" when it comes to western blotting substrates. With so many options on the market, it is sometimes difficult to determine which substrate performs the best for individual western blot systems. In this webinar we will discuss the best methods for optimizing this technique as well as how to determine the appropriate reagents for your sensitive target detection.

Webinar

Protein concentration assays – novel, simplified and rapid techniques for quantifying proteins in solution

Speaker:

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

Biography:

Ramesh Ganapathy joined Thermo Fisher Scientific in 2001 after completing his Ph.D. at University of Wisconsin-Madison and a post-doctoral fellowship at the Department of Bioengineering at 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 ranging from resins, substrates for western blotting, magnetic beads, and peptide and protein assay products.

Abstract

Protein concentration assays are a widely used technique in protein research to determine the total amount of protein in a sample prior to downstream applications.  While there are many protein assay options available for scientists, it is important to choose one that delivers the most accurate results based on the sample type, protein preparation methodologies and downstream applications. New developments and a recently introduced product in this area now provide scientists the full combination of quality and ease-of-use by offering high accuracy and reproducibility. In this webinar, we will discuss the principles behind the most popular protein concentration assays, how to achieve the highest quality results with your sample, and the importance of using the proper standards, protein preparation, sample composition, and downstream applications in choosing the right protein assay for your needs.

Learning objectives:

  • Choose the best colorimetric protein assay based on sample composition.
  • Understand the differences between dye-based (Bradford, Pierce 660 nm assay) and Copper-chelating (BCA) assays.
  • Describe the different instruments used with protein assays

Webinar

Harnessing the power of TMT11plex sample multiplexing and improved phosphopeptide enrichment to gain new insights into signaling pathways

Speaker:

Ryan Bomgarden, Ph.D.
Senior R&D Staff Scientist, Protein and Cell Analysis, Thermo Fisher Scientific

Biography:

Ryan Bomgarden received his bachelor’s degree from Coe College with majors in chemistry and molecular biology. He received his Ph.D. at Stanford University under Dr. Karlene Cimprich where he worked on biochemical characterization of the checkpoint kinase ATR DNA binding activity and signaling in UV-sensitive human cell lines. Since joining Thermo Fisher Scientific in protein biology R&D, he has worked on the development of key protein research reagents including photo-reactive crosslinkers, SILAC metabolic labeling kits and subcellular protein fractionation kits. Currently Dr. Bomgarden is in the mass spectrometry R&D group where his development work includes tandem Mass Tag reagents for relative quantitation of proteomic samples, HeLa protein digest standards for LC-MS QC, MS-cleaveable crosslinkers for protein structure analysis, heavy protein IVT kits for production of stable isotope-labeled proteins; and Thermo Scientific ActivX probes for kinase, GTPase and serine hydrolase inhibitor profiling and detection. In addition to his work at Thermo Fisher Scientific, Dr. Bomgarden is also an adjunct professor at the University of Illinois College of Medicine where he teaches courses on biochemical techniques, drug design and ethics. Dr. Bomgarden is also heavily involved in STEM education, creating and performing science demonstrations for local schools and non-profit organizations.

Abstract

Mass spectrometry (MS) has become the predominant technology to analyze proteins due to its ability to identify and characterize proteins and their modifications with high sensitivity and selectivity.  Increasing the number of samples analyzed simultaneously in a single mass spectrometry experiment is essential for increased sample throughput, fewer missing measurements between samples, and increased statistical power among replicates.  Thermo Scientific Tandem Mass Tag TMT Reagents are one technology which enables concurrent identification and multiplexed quantitation of different samples using tandem mass spectrometry. Recently, we have extended the multiplexing capabilities of TMT Reagents from 6 to 11 without an increase in the tag size or structure by utilizing the mass difference between 13C and 15N isotopes in the reporter region.  We have also developed new phosphopeptide enrichment workflows with improved phosphopeptide yield, selectivity and identification rates.  Overall, these additions to the TMT workflow enable higher sample multiplexing and provide in depth quantification of protein post-translational modifications.

Learning objectives:

  • Understand the basics of how tandem mass tags can be used for protein multiplexing 
  • Discuss how the multiplexing capabilities of TMT Reagents were extended from 6 to 11 without an increase in the tag size or structure 
  • Identify the differences between Fe-NTA and TiO2 and how to use sequentially for maximum phosphopeptide enrichment

Webinar

Strategies for successful crosslinking and bioconjugation applications

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 significant impact on the bioscience fields as proven by nearly 27,500 citations to his publications and patents.

Abstract

Crosslinking and bioconjugation reagents are widely used in research to study protein interactions, or as tools for assay or nanoparticle development. While there are many reactive chemistries and labeling tags available to scientists, it is important to choose the tools that deliver the desired outcome based on the functional groups available, steric considerations, cleavability and solubility required for successful conjugation. In this webinar, we will discuss the basics of crosslinking, and simple guidelines on how to achieve the most efficient modification for some typical applications, including labeling antibodies, immobilizing biomolecules to surfaces, and capturing protein interactions for mass spectrometry analysis.