3rd Annual 7 Steps of Protein Virtual Event

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

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

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

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 a 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

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 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

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), incorrect reducing agent, 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

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

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

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

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

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).

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

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.

• 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

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

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.

Western blotting是科研工作者广泛使用的一种技术，用于检测细胞或者组织裂解液中的蛋白表达情况。本次讲座将会讲述western blotting的流程，包括电泳、转膜、检测三大模块；同时介绍我们公司能为客户提供的western blotting全流程的解决方案，以及western blotting相关小贴士，目的帮助客户更加有效和高质量的获得western blotting结果。

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
• 掌握western blotting的整个流程
• 了解赛默飞western blotting的相关工具
• 获得完美western blotting结果的小贴士