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Introduction to Karl Fischer Titration

Andrea Felgner, Hydranal Market Segment Manager
As a leading authority on Karl Fischer Titration, we have extensive experience of product development and customer applications.
Karl Fischer Titration is generally the most accepted method of water content determination and the webinar will provide an introduction to the technique.
May 15 2012
56 mins
Introduction to Karl Fischer Titration
  • Channel
  • Channel profile
  • Interplay of Acetyltransferase EP300 and the Proteasome System in Regulat-Sess.2 May 21 2015 8:00 pm UTC 75 mins
    Mirko Theis & Christian Loew
    In the present study, a genome-wide RNA interference screen was combined with an extensive biochemical analysis and quantitative proteomics to better understand the regulation of the heat-shock response (HSR) upon thermal stress. The usage of an endoribonuclease-prepared small interfering RNA (esiRNA) library represented a simple and efficient way to perform RNAi with minimal off-target effects. In the screening experiments novel positive and negative modulators of the stress response were identified, including proteins involved in chromatin remodeling, transcription, mRNA splicing, DNA damage repair, and proteolytic degradation. The diversity of the identified regulators suggests that induction and attenuation of the HSR integrate signals from different cellular pathways and are rather multi-factorial processes than single gene/protein events. The modulator proteins are localized in multiple cellular compartments with the majority having their primary location in the nucleus. A protein-protein interaction analysis revealed a HSR regulatory network, with chromatin modifiers and nuclear protein quality control components occupying hub positions. These observations are supported by quantitative proteomics experiments, which showed specific stress-induced reorganizations of the nuclear proteome, including the transient accumulation of chaperones and proteasomal subunits.
    Moreover, we found that the acetyltransferase EP300 controls the cellular level of activatable HSF1. This involves acetylation of HSF1 at multiple lysines not required for function and results in stabilization of HSF1 against proteasomal turnover. Acetylation of functionally critical lysines during stress serves to fine-tune HSF1 activation. Finally, the nuclear proteasome system functions in attenuating the stress response by degrading activated HSF1 in a manner linked with the clearance of misfolded proteins.
  • Interplay of Acetyltransferase EP300 and the Proteasome System in Regu-Session 1 May 21 2015 12:00 pm UTC 75 mins
    Mirko Theis & Christian Loew
    In the present study, a genome-wide RNA interference screen was combined with an extensive biochemical analysis and quantitative proteomics to better understand the regulation of the heat-shock response (HSR) upon thermal stress. The usage of an endoribonuclease-prepared small interfering RNA (esiRNA) library represented a simple and efficient way to perform RNAi with minimal off-target effects. In the screening experiments novel positive and negative modulators of the stress response were identified, including proteins involved in chromatin remodeling, transcription, mRNA splicing, DNA damage repair, and proteolytic degradation. The diversity of the identified regulators suggests that induction and attenuation of the HSR integrate signals from different cellular pathways and are rather multi-factorial processes than single gene/protein events. The modulator proteins are localized in multiple cellular compartments with the majority having their primary location in the nucleus. A protein-protein interaction analysis revealed a HSR regulatory network, with chromatin modifiers and nuclear protein quality control components occupying hub positions. These observations are supported by quantitative proteomics experiments, which showed specific stress-induced reorganizations of the nuclear proteome, including the transient accumulation of chaperones and proteasomal subunits.
    Moreover, we found that the acetyltransferase EP300 controls the cellular level of activatable HSF1. This involves acetylation of HSF1 at multiple lysines not required for function and results in stabilization of HSF1 against proteasomal turnover. Acetylation of functionally critical lysines during stress serves to fine-tune HSF1 activation. Finally, the nuclear proteasome system functions in attenuating the stress response by degrading activated HSF1 in a manner linked with the clearance of misfolded proteins.
  • Sample Preparation Considerations for Multiplexed MRM LC-MS Protein Assays. May 18 2015 3:00 pm UTC 75 mins
    Kevin Ray,Ph.D. Sr Manager-Analytical R&D,S-A & Steve Hunsucker,Ph.D. Sr Director-Lab Operations, Integrated Diagnostics Inc
    Multiplexed protein assays have tremendous potential in clinical diagnostics, in particular measurement of proteins in plasma or serum derived from circulating blood. The dynamic range of protein concentration in these samples, and the dominance of very high abundance proteins such as albumin and immunoglobulins, make measurement of low concentration proteins impossible without some type of enrichment approach. This workshop will discuss the benefits of using Seppro® protein depletion columns in sample preparation for multiplexed LC-MS protein clinical assays.
  • Sample Preparation Considerations for Multiplexed MRM LC-MS Protein Assays May 18 2015 1:00 am UTC 75 mins
    Kevin Ray,Ph.D. Sr Manager-Analytical R&D,S-A & Steve Hunsucker,Ph.D. Snr Director-Lab Operations, Integrated Diagnostics Inc
    Multiplexed protein assays have tremendous potential in clinical diagnostics, in particular measurement of proteins in plasma or serum derived from circulating blood. The dynamic range of protein concentration in these samples, and the dominance of very high abundance proteins such as albumin and immunoglobulins, make measurement of low concentration proteins impossible without some type of enrichment approach. This workshop will discuss the benefits of using Seppro® protein depletion columns in sample preparation for multiplexed LC-MS protein clinical assays.
  • Duolink in Biomedical Science, Session 2 Apr 28 2015 3:00 pm UTC 75 mins
    J. Matthew Rhett, PhD Instructor, Department of Surgery, Medical University of South Carolina
    Gap junctions (GJs) are large aggregates of intercellular channels that facilitate the diffusion of small molecules and ions between two interacting cells. GJ intercellular channels are formed through the interaction of two half-channels, called hemichannels, composed of oligomerized connexin protein subunits. Both GJs and hemichannels have numerous important physiological and pathological roles in tissue functions including propagation of the action potential in the heart, tumor growth and metastasis, the inflammatory response and adaptive immunity, wound healing, and electrical synaptic transmission in the central nervous system. The most widely expressed connexin isoform is Cx43, and its regulation in the abovementioned processes has been a major focus of GJ research. Over the past two decades protein-protein interaction with the cytoplasmic carboxyl terminus of Cx43 has come to the fore as an endogenous mechanism for controlling the GJ life cycle, channel gating, and channel-independent functions. We have used the Duolink proximity ligation assay (PLA) as a technique to study protein interactions with Cx43 in cultured cells. Two unique aspects of the technology – specifically, subcellular localization and the binary nature of the labeling – in combination with standard immunofluorescent confocal imaging techniques have yielded unexpected insights into GJ ultrastructure, action potential conduction, and the mechanistic regulation of Cx43 trafficking and hemichannel accretion to GJ plaques. In this context, the practical application of, appropriate controls for, and interpretation of Duolink PLAs will be explicated.
  • Practice-Duolink in Biomedical Science, Session 1 Apr 28 2015 1:00 am UTC 75 mins
    J. Matthew Rhett, PhD Instructor, Department of Surgery, Medical University of South Carolina
    Gap junctions (GJs) are large aggregates of intercellular channels that facilitate the diffusion of small molecules and ions between two interacting cells. GJ intercellular channels are formed through the interaction of two half-channels, called hemichannels, composed of oligomerized connexin protein subunits. Both GJs and hemichannels have numerous important physiological and pathological roles in tissue functions including propagation of the action potential in the heart, tumor growth and metastasis, the inflammatory response and adaptive immunity, wound healing, and electrical synaptic transmission in the central nervous system. The most widely expressed connexin isoform is Cx43, and its regulation in the abovementioned processes has been a major focus of GJ research. Over the past two decades protein-protein interaction with the cytoplasmic carboxyl terminus of Cx43 has come to the fore as an endogenous mechanism for controlling the GJ life cycle, channel gating, and channel-independent functions. We have used the Duolink proximity ligation assay (PLA) as a technique to study protein interactions with Cx43 in cultured cells. Two unique aspects of the technology – specifically, subcellular localization and the binary nature of the labeling – in combination with standard immunofluorescent confocal imaging techniques have yielded unexpected insights into GJ ultrastructure, action potential conduction, and the mechanistic regulation of Cx43 trafficking and hemichannel accretion to GJ plaques. In this context, the practical application of, appropriate controls for, and interpretation of Duolink PLAs will be explicated.
  • Visualizing DNA Methylation (5mC) on a Specific Genomic Locus (SEPTIN9) 02 Recorded: Apr 14 2015 37 mins
    Dr. Vikas Palhan, Sr. Scientist, Molecular Biotechnology, Sigma-Aldrich
    Although there are many techniques to study epigenetic marks such as DNA- and histone-methylation, on a genomic scale, there exists a need in the field to visualize these epigenetic marks at a single genomic locus in individual cells. Such an application requires a highly sensitive detection method. With this aim, a protocol was developed to perform in-situ hybridization followed by proximity ligation assay (a.k.a. Duolink®) and cell imaging to visualize DNA-methylation (5meC) on the SEPTIN9 promoter. SEPTIN9 promoter methylation is a known biomarker for colon cancer. After optimizing cross-linking, cell permeabilization and chromatin accessibility, the genomic specificity was ascertained by hybridizing with a pool of biotinylated-oligo probes that target the CpG islands in the human SEPTIN9 promoter. The Duolink assay was performed using anti-biotin and anti-5meC antibodies, corresponding proximity ligation assay probes, and Far Red detection reagents.
    Imaging by fluorescent microscopy revealed two red punctate spots in metastatic prostate cancer DU145 cells (diploid for chromosome 17 – location of SEPTIN9 gene) and three red spots in colon cancer SW480 cells (triploid for Chr17). No signal was observed in normal cells (BJ) or with non-specific oligo probes (LacZ). A decrease in Duolink signal was observed when the DU145 cells were treated with 5-AzaC, a drug known to block DNA-methylation. This proof of concept study will be extended to frozen and formalin fixed paraffin embedded human cancer tissue samples.
    Fluorescent imaging data will also be presented from a Duolink assay to monitor the interaction of EZH2 histone methyltransferase with the H3K27me3 epigenetic mark in prostate cancer (DU145) cells. Reduction in the Duolink signal demonstrated inhibition of EZH2 activity by the small molecule inhibitors SAHA and GSK343.
  • Visualizing DNA Methylation (5mC) on a Specific Genomic Locus (SEPTIN9) 01 Recorded: Apr 14 2015 33 mins
    Dr. Vikas Palhan, Sr. Scientist, Molecular Biotechnology, Sigma-Aldrich
    Although there are many techniques to study epigenetic marks such as DNA- and histone-methylation, on a genomic scale, there exists a need in the field to visualize these epigenetic marks at a single genomic locus in individual cells. Such an application requires a highly sensitive detection method. With this aim, a protocol was developed to perform in-situ hybridization followed by proximity ligation assay (a.k.a. Duolink®) and cell imaging to visualize DNA-methylation (5meC) on the SEPTIN9 promoter. SEPTIN9 promoter methylation is a known biomarker for colon cancer. After optimizing cross-linking, cell permeabilization and chromatin accessibility, the genomic specificity was ascertained by hybridizing with a pool of biotinylated-oligo probes that target the CpG islands in the human SEPTIN9 promoter. The Duolink assay was performed using anti-biotin and anti-5meC antibodies, corresponding proximity ligation assay probes, and Far Red detection reagents.
    Imaging by fluorescent microscopy revealed two red punctate spots in metastatic prostate cancer DU145 cells (diploid for chromosome 17 – location of SEPTIN9 gene) and three red spots in colon cancer SW480 cells (triploid for Chr17). No signal was observed in normal cells (BJ) or with non-specific oligo probes (LacZ). A decrease in Duolink signal was observed when the DU145 cells were treated with 5-AzaC, a drug known to block DNA-methylation. This proof of concept study will be extended to frozen and formalin fixed paraffin embedded human cancer tissue samples.
    Fluorescent imaging data will also be presented from a Duolink assay to monitor the interaction of EZH2 histone methyltransferase with the H3K27me3 epigenetic mark in prostate cancer (DU145) cells. Reduction in the Duolink signal demonstrated inhibition of EZH2 activity by the small molecule inhibitors SAHA and GSK343.
  • Late Stage Functionalization of Drug-Like Molecules Session 2 Recorded: Mar 31 2015 62 mins
    Tim Cernak
    C-H functionalization is a powerful addition to the toolbox of the medicinal chemist. Modern C-H functionalization techniques hold the potential to enable the rapid exploration of structure activity relationships (SAR), the generation of oxidized metabolites, the blocking of metabolic hot spots and the preparation of biological probes. This presentation will describe a variety of high-value C-H functionalization chemistries, developed in house and in collaboration with academic groups, and give examples of how these technologies have been deployed successfully to impact drug discovery programs.
  • Why We Use Cell Lines as Models: A 30 Year Journey and What Lies Ahead Recorded: Mar 26 2015 53 mins
    Jim Cooper, Cell Biology Application Scientist, Scientific Development Group, ECACC
    This webinar will discuss the evolving role of Cell Lines and Cell Culture and their usefulness as models of physiological function and disease. The webinar will review the depth, breadth and best use of currently available Cell Lines. The role played by Cell Lines in ensuring that the output of future research continues to be valid and credible will also be evaluated. This is the first of a three part webinar series focused on delivering a broad picture of the role of cell lines as models in scientific research, the various strategies and approaches available.
  • Molecular Annotation of EGFR Signaling-Associated Complexes in Human Cancer 02 Recorded: Mar 26 2015 53 mins
    Eric B. Haura M.D., Moffitt Cancer Center
    Mass spectrometry and other methods such as yeast two-hybrid can now accurately discern protein complexes or larger protein interactomes in diseases such as cancer, yet it is difficult to forward translate this knowledge into human samples. To overcome this hurdle, we began experiments using proximity ligation assays (PLA) to directly translate protein complexes into human tumor materials. Our assay reflects protein complexes between EGFR and GRB2 protein, a key adaptor protein necessary for EGFR pathway activation and coupling to downstream MAPK signaling. We annotated nearly 300 primary xenograft models (PDX) of cancer and show tumor subtype enrichment of EGFR:GRB2 signaling-associated complexes. Furthermore, tumors with abundant levels of EGFR:GRB2 signaling-associated complexes are more likely to respond to anti-EGFR antibody-based therapy. Finally, in 350 lung cancer tissues, across three distinct cohorts of patients, we demonstrate the ability of EGFR:GRB2 protein complexes to segregate tumors and show benefit to EGFR tyrosine kinase inhibitor therapy in patients whose tumors harbor high levels of EGFR:GRB2 signaling-associated complexes. This suggests that annotation of signaling-associated protein complexes in cancer tissues can not only molecularly annotate disease types but may also have predictive capacity for cancer therapeutics. This work opens up the human protein interactome as a new class of molecular markers for disease in a more practical manner. Proteins, encoded by DNA, do not work in isolation but instead function as part of multi-protein complexes that drive both normal and disease physiology. While we demonstrate the utility of this approach in cancer, receptor tyrosine kinase signaling and tyrosine kinase inhibitor therapeutics, our approach described here could have utility across a wide spectrum of both signaling-associated complexes and different types of disease, and thus would be attractive to a large audience.
  • Why We Use Cell Lines as Models: A 30 Year Journey and What Lies Ahead Recorded: Mar 26 2015 60 mins
    Jim Cooper, Cell Biology Application Scientist, Scientific Development Group, ECACC
    This webinar will discuss the evolving role of Cell Lines and Cell Culture and their usefulness as models of physiological function and disease. The webinar will review the depth, breadth and best use of currently available Cell Lines. The role played by Cell Lines in ensuring that the output of future research continues to be valid and credible will also be evaluated. This is the first of a three part webinar series focused on delivering a broad picture of the role of cell lines as models in scientific research, the various strategies and approaches available.
  • Molecular Annotation of EGFR Signaling-Associated Complexes in Human Cancer 01 Recorded: Mar 26 2015 50 mins
    Eric B. Haura M.D., Moffitt Cancer Center
    Mass spectrometry and other methods such as yeast two-hybrid can now accurately discern protein complexes or larger protein interactomes in diseases such as cancer, yet it is difficult to forward translate this knowledge into human samples. To overcome this hurdle, we began experiments using proximity ligation assays (PLA) to directly translate protein complexes into human tumor materials. Our assay reflects protein complexes between EGFR and GRB2 protein, a key adaptor protein necessary for EGFR pathway activation and coupling to downstream MAPK signaling. We annotated nearly 300 primary xenograft models (PDX) of cancer and show tumor subtype enrichment of EGFR:GRB2 signaling-associated complexes. Furthermore, tumors with abundant levels of EGFR:GRB2 signaling-associated complexes are more likely to respond to anti-EGFR antibody-based therapy. Finally, in 350 lung cancer tissues, across three distinct cohorts of patients, we demonstrate the ability of EGFR:GRB2 protein complexes to segregate tumors and show benefit to EGFR tyrosine kinase inhibitor therapy in patients whose tumors harbor high levels of EGFR:GRB2 signaling-associated complexes. This suggests that annotation of signaling-associated protein complexes in cancer tissues can not only molecularly annotate disease types but may also have predictive capacity for cancer therapeutics. This work opens up the human protein interactome as a new class of molecular markers for disease in a more practical manner. Proteins, encoded by DNA, do not work in isolation but instead function as part of multi-protein complexes that drive both normal and disease physiology. While we demonstrate the utility of this approach in cancer, receptor tyrosine kinase signaling and tyrosine kinase inhibitor therapeutics, our approach described here could have utility across a wide spectrum of both signaling-associated complexes and different types of disease, and thus would be attractive to a large audience.
  • Integrating the Proximity Ligation Assay (PLA) into your Research Program 02 Recorded: Mar 17 2015 49 mins
    Joe Roethele Director of Antibody Production, Bethyl Laboratories
    The proximity ligation assay (PLA) is a powerful technique for performing in situ analysis of antibody targets in cells or tissues. This antibody-based application is very robust, and will provide unique data on localization and/or interaction of proteins. The webinar will outline the development of PLA, using an approach that is designed to help all researchers—regardless of their level of experience with this application. Topics addressed will include the following: guidelines for getting started, tips on the crucial bioinformatics investigations involved with the planning phase, specific details on the bench-work associated with performing the assay, and troubleshooting suggestions to allow investigators to refine the assay and successfully integrate it into their research program.
  • Integrating the Proximity Ligation Assay (PLA) into your Research Program 01 Recorded: Mar 17 2015 51 mins
    Joe Roethele Director of Antibody Production, Bethyl Laboratories
    The proximity ligation assay (PLA) is a powerful technique for performing in situ analysis of antibody targets in cells or tissues. This antibody-based application is very robust, and will provide unique data on localization and/or interaction of proteins. The webinar will outline the development of PLA, using an approach that is designed to help all researchers—regardless of their level of experience with this application. Topics addressed will include the following: guidelines for getting started, tips on the crucial bioinformatics investigations involved with the planning phase, specific details on the bench-work associated with performing the assay, and troubleshooting suggestions to allow investigators to refine the assay and successfully integrate it into their research program.
  • Introduction to PLA and Duolink Session 2 Recorded: Mar 10 2015 46 mins
    Dr. Ola Söderberg, Uppsala University and Tom Juehne, Sigma-Aldrich
    Visualization of protein-protein interactions at endogenous levels of expression is a powerful advancement in Life Science. Duolink® is a versatile tool for detection, quantification and localization of cytoplasmic signaling events. PLA® provides the ability to see and quantitate protein posttranslational modifications, define complex interactions, illuminate complex protein clusters or amplify low abundant single events.
  • Introduction to PLA and Duolink Session 1 Recorded: Mar 10 2015 32 mins
    Dr. Ola Söderberg, Uppsala University and Tom Juehne, Sigma-Aldrich
    Visualization of protein-protein interactions at endogenous levels of expression is a powerful advancement in Life Science. Duolink® is a versatile tool for detection, quantification and localization of cytoplasmic signaling events. PLA® provides the ability to see and quantitate protein posttranslational modifications, define complex interactions, illuminate complex protein clusters or amplify low abundant single events.
  • Modifying Cell Lines for Target Discovery and Validation Recorded: Mar 3 2015 27 mins
    Ethan S. Patterson, PhD
    Recent advances in genome editing have opened new potential for a wide variety of specific genomic modifications in virtually any cell type. In this 30-minute seminar, we will discuss the process of genome editing, address factors affecting the design of a genome editing project, and explore the application of modified cell lines for target discovery and validation.
  • Purification & Workup of Organic Synthesis - Basics, Strategies & Methodology Recorded: Nov 6 2014 68 mins
    Jason Wrigley, Scientific Liaison Team - Analytical Chemistry (Sigma-Aldrich)
    Workup and purification can be a complicated and time consuming process. The webinar will offer some available options for streamlining this part of your workflow, and provide some guidance and strategic advice depending on budget, equipment and experience. Workup of aqueous fractions will also be discussed.

    With the need for results as quickly as possible we will discuss how to perform reaction monitoring as quickly as possible using standard HPLC systems. We will show, with an example, how you may be able to obtain your results in 1/3 of your current time if you are running isocratically, or with generic gradients, with 5um or 3um C18 HPLC columns.
  • 3D Cell Culture Innovations Session 2 Recorded: Nov 4 2014 58 mins
    Dr. Nicky Slawny Applications Director 3D Biomatrix Dr. John Dangerfield Chief Operating Officer Austrianova
    This 1 hour long webinar will cover innovations in 3D cell culture technologies. Technology experts will discuss the unique features of the Perfecta3D Hanging Drop Plates and Cell-in-a-Box live cell encapsulation system with special emphasis on research applications and techniques.

    Topics will include:
    •Overview of the Perfecta3D plates and Cell-in-a-Box system
    •3D culture system selection
    •Best practices
    •Research Applications
Focusing on new / innovative technologies and industry challenges
The Sigma-Aldrich seminar series features scientific presentations from key specialists in analytical chemistry, chemistry and life sciences, on the practical and technical aspects of new developments and innovations, to help advance your research.

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  • Title: Introduction to Karl Fischer Titration
  • Live at: May 15 2012 1:00 pm
  • Presented by: Andrea Felgner, Hydranal Market Segment Manager
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