Protocol
Next-generation MALDI top-down sequencing of protein biotherapeutics – expanding the scope of timsTOF technology
This new method takes advantage of Trapped Ion Mobility Spectrometry (TIMS), to further enhance MALDI-TDS by adding another dimension of separation in the gas phase.High confidence N- and C-terminal sequencing specifically are enhanced by combining high resolution (~ 60,000 RP) and 2 ppm mass accura... More
Protocol
Achieving Reproducible and Automated Region-Specific Lipid Annotations using CCS-aware SpatialOMx®
The CCS-aware SpatialOMx® workflow opens new dimensions by combining the molecular and spatial information measured by MALDI-TIMS Imaging with highly confident 4D-Omics annotations. MetaboScape® 2021b and SCiLS™ Lab 2021a provide the interface to match data from both ionization techniques and enable... More
Protocol
How to Determine Glycan Profiles of Biopharmaceuticals from Peptide Mapping Data
Glycosylation is a common critical quality attribute (CQA) of therapeutic proteins and needs to be characterized during product development. Typically, this analysis is conducted after enzymatic release and tagging of the glycans followed by fluorescence and MS detection. However, that approach lose... More
Protocol
High throughput 4D-Proteomics – Application of dia-PASEF® and the Evosep One for short gradients
In this application note, we demonstrate the benefits of dia-PASEF technology on the timsTOF Pro platform coupled to an Evosep One for high-throughput in-depth proteome analysis of up to 300 samples per day (SPD). We quantify about 5200 protein groups in only 21-minutes run time. In 4.8-minutes ultr... More
Protocol
Elucidation of metabolic changes in HFD-ApoE–/– model by SP6 peptide: A flow injection analysis magnetic resonance mass spectrometry (FIA-MRMS) study
The employment of natural peptides in the treatment of cardiovascular diseases is an attractive option to pharmacological therapy. In this study, a novel antihypertensive peptide from Spirulina Platensis (SP6) was employed to evaluate its potential anti-atherosclerotic effect in high fat diet ApoE–/... More
Protocol
Magnetic Resonance Mass Spectrometry (MRMS) discriminates yeast mutants through metabolomics
A eukaryote model of Saccharomyces cerevisiae was used to study the methylglyoxal pathway by Magnetic resonance mass spectrometry (MRMS). It was discovered that glutathione plays a major role in driving metabolomic differences between different strains. It is expected that metabolomics-based discrim... More
Protocol
Proteomic Interrogation of Primary Insulin-secreting Pancreatic β-cells
In this work we leveraged the analytical performance of the timsTOF Pro to identify expression of low-level enzymes for the first time in primary insulinsecreting beta cells (β-cells). We found that these proteins are critical, functional components of key metabolic pathways which can support β-cell... More
Protocol
Studying protein-drug complexes under native conditions in the low nM range using MRMS
Protein ligand complexes need to be preserved through the electrospray and detection process. This work describes high resolution native mass spectrometric measurements of the enzyme carbonic anhydrase II (Figure 1) and the non-covalent complex with the substrate acetazolamide (Figure 2) using a sci... More
Protocol
Incorporating CCS values to enable 4-dimensional annotation of metabolic features
Reliable annotation of metabolites for LC-MS/MS based data requires the adept combination of many parameters such as retention time and accurate mass. To extend our parameter portfolio, we analyzed the high reproducible measured CCS values using trapped ion mobility spectrometry (TIMS) to provide ad... More
Protocol
prm-PASEF®: enabling high-throughput, high sensitivity targeted proteomics
In comparison with standard selected and parallel reaction monitoring (SRM and PRM), the prm-PASEF increases the number of peptides that can be targeted in a single acquisition method, without compromising the selectivity or the sensitivity. Using a prototype acquisition software, we targeted 201 is... More
Protocol
Long Term Storage, Recycling and Simplified Calcium Phosphate Transfection Protocols for Cell Cultures
Long term storage of cells is vital for use in the future experiments. Current methods of storing cells under liquid nitrogen vapor is costly, and requires regular monitoring and filling of the storage tank. We stored several cell lines in -80oC freezer over a decade and revived them successfully. S... More
Protocol
Zebrafish Tumor Graft Transplantation to Grow Tumors In Vivo That Engraft Poorly as Single Cell Suspensions
Angiosarcoma is a clinically aggressive tumor with a high rate of mortality. It can arise in vascular or lymphatic tissues, involve any part of the body, and aggressively spread locally or metastasize. Angiosarcomas spontaneously develop in the tp53 deleted (tp53del/del) zebrafish mutant. However, e... More
Protocol
An Automated Low-Cost Swim Tunnel for Measuring Swimming Performance in Fish
The study of swimming behavior is an important part of fish biology research and the swim tunnel is used to study swimming performance as well as metabolism of fish. In this investigation, we have developed a user-friendly, automated, modular, and low-cost swim tunnel that permits to study the perfo... More
Protocol
A Rabbit Femoral Condyle Defect Model for Assessment of Osteochondral Tissue Regeneration
Osteochondral tissue repair represents a common clinical need, with multiple approaches in tissue engineering and regenerative medicine being investigated for the repair of defects of articular cartilage and subchondral bone. A full thickness rabbit femoral condyle defect is a clinically relevant mo... More
Protocol
Cartilage Assessment Requires a Surface Characterization Protocol: Roughness, Friction, and Function
The surface of articular cartilage is integral to smooth, low-friction joint articulation. However, the majority of cartilage literature rarely includes measurements of surface characteristics and function. This may, in part, be due to a shortage of or unfamiliarity with fast, nondestructive, and, p... More
Protocol
Optimized Protocol for Accurate Titration of Adeno-Associated Virus Vectors
Adeno-associated virus (AAV) is currently the most popular gene delivery vector for in vivo gene therapy. However, variability in titration methods between different laboratories affects the reproducibility of experiments and evaluation of safety and efficacy in clinical trials. We describe an optim... More
Protocol
Single-Use Capture Purification of Adeno-Associated Viral Gene Transfer Vectors by Membrane-Based Steric Exclusion Chromatography
We present membrane-based steric exclusion chromatography (SXC) as a universal capture step for purification of adeno-associated virus (AAV) gene transfer vectors independent of their serotype and surface characteristics. SXC is performed by mixing an unpurified cell culture supernatant containing A... More
Protocol
Development and Validation of an Anion Exchange High-Performance Liquid Chromatography Method for Analysis of Empty Capsids and Capsids Encapsidating Genetic Material in a Purified Preparation of Recombinant Adeno-Associated Virus Serotype 5
The development of various manufacturing platforms and analytical technologies has substantially contributed to successfully translating the recombinant adeno-associated viral vector from the laboratory to the clinic. The active deployment of these analytical technologies for process and product cha... More
Protocol
Predictive Low-Glucose Suspend Necessitates Less Carbohydrate Supplementation to Rescue Hypoglycemia: Need to Revisit Current Hypoglycemia Treatment Guidelines
Current guidelines recommend 15–20 g of carbohydrate (CHO) for treatment of mild to moderate hypoglycemia. However, these guidelines do not account for reduced insulin during suspensions with predictive low-glucose suspend (PLGS). We assessed insulin suspensions, hypoglycemic events, and CHO treatme... More
Protocol
Insulin Metrics: Need for Development of Consensus Standards for Reporting of Insulin Dosing Data
The advent of connected insulin pens will generate an avalanche of digital insulin data, especially in the context of prandial- and multiple daily injection-insulin regimens. There is a need for the diabetes community to develop standards for such data, analogous to what has been achieved using the ... More
Application
Knock-out cell lines and their use in signaling pathways
Knock-out (KO) cell lines are powerful tools to study disease mechanisms, such as the effect of gene disruption on downstream pathways. In this application note, we highlight the effectiveness of Abcam KO cell lines that can be used to investigate signaling pathways, in this case, the TRAIL pathway.... More
Application
Knock-out cell lines and their use in signaling pathways
Knock-out (KO) cell lines are powerful tools to study disease mechanisms, such as the effect of gene disruption on downstream pathways. In this application note, we highlight the effectiveness of Abcam KO cell lines that can be used to investigate signaling pathways, in this case, the TRAIL pathway.... More
Application
Recombinant RabMAb®: Reliable antibodies for reproducible results
Poorly characterized antibodies often fail to provide consistent, reproducible results, thereby wasting materials, researcher time, and money. Compared to traditional polyclonal and monoclonal antibodies, recombinant antibodies exhibit very little batch-to-batch variability, providing scientists wit... More
Application
Recombinant RabMAb®: Reliable antibodies for reproducible results
Poorly characterized antibodies often fail to provide consistent, reproducible results, thereby wasting materials, researcher time, and money. Compared to traditional polyclonal and monoclonal antibodies, recombinant antibodies exhibit very little batch-to-batch variability, providing scientists wit... More
Application
Antibody conjugation guide
Antibody conjugation, also known as antibody labeling, is the process of chemically linking an antibody to a specific tag. There are many common methods used to conjugate antibodies, although they can have complex protocols and usually require a certain level of skill and expertise. In this guide, w... More
Application
Antibody conjugation guide
Antibody conjugation, also known as antibody labeling, is the process of chemically linking an antibody to a specific tag. There are many common methods used to conjugate antibodies, although they can have complex protocols and usually require a certain level of skill and expertise. In this guide, w... More
Application
Batch-to-batch consistency of premium grade bioactive proteins
This white paper demonstrates the batch-to-batch consistency of our premium grade bioactive proteins (growth factors and cytokines). Designed to meet the exacting demands of cell and gene therapy applications, our premium grade proteins are ideal for controlled cell culture. ... More
Application
Batch-to-batch consistency of premium grade bioactive proteins
This white paper demonstrates the batch-to-batch consistency of our premium grade bioactive proteins (growth factors and cytokines). Designed to meet the exacting demands of cell and gene therapy applications, our premium grade proteins are ideal for controlled cell culture. ... More
Application
Technical tips for ELISA and multiplex immunoassay development
Developing a high performance sandwich ELISA or multiplex immunoassay can be complicated, and high performance requires careful development and validation. To help, we have created this guide based on what we have learned during the development of over 500 matched antibody pairs, ELISA kits, and mul... More
Application
Technical tips for ELISA and multiplex immunoassay development
Developing a high performance sandwich ELISA or multiplex immunoassay can be complicated, and high performance requires careful development and validation. To help, we have created this guide based on what we have learned during the development of over 500 matched antibody pairs, ELISA kits, and mul... More
Primer
AI-Powered Phenotypic Profiling Revolutionizes Drug Discovery
Today, nine out of ten drugs fail in clinical trials. Furthermore, it takes over a decade and an average cost of $2 billion to develop and approve each medicine.1 An important underlying reason is the gap that exists between cell-based in vitro research and clinical research—often referred to as the... More
Primer
Bio-Electrospraying and Cell Electrospinning: A Sea of Applications
Electrospraying and electrospinning have been used for a wide range of applications ranging from coatings, propulsion, and pesticide sprays to scaffolding a wide range of advanced materials for applications in filtration and tissue engineering. Although electrospun scaffolds have been explored as ti... More
Primer
Quick Antibiotic Susceptibility Test for High-Threat Pathogens in Whole Blood Samples
By spreading so rapidly and wreaking so much havoc, COVID-19 has revealed the susceptibility of global societies to both natural pathogens and intentionally released bioterror agents. The latter include bacteria that have been engineered for enhanced antibiotic resistance.1 Once pathogenic bacteria ... More
Primer
Impedance Spectroscopy Measures Antimicrobial Susceptibility in Real Time
Antimicrobial drugs save millions of lives each year. However, in 2019, antibiotic-resistant microorganisms were associated with nearly 5 million deaths worldwide1. The alarming trend of bacterial antimicrobial resistance may further worsen as many COVID-19 patients are administered broad spectrum a... More
Primer
Methods to Improve Efficiency of Antimicrobial Susceptibility Testing: Microfluidic Impedance Cytometry
The global issue of antimicrobial resistance has been called a “slow motion catastrophe” resulting from the excessive and often inappropriate use of antibiotics to fight bacterial infections.1 A long-standing unmet need is the availability of a quick and easy clinical test to support a more evidence... More
Primer
Tips for Increasing Transfection Efficiency
Chemical transfection, which relies on the formation of a condensed complex of positively chargedliposomal or non-liposomal reagents and negatively charged nucleic acids,is the most common method for delivering foreign genetic material into cultured cells. Complexes are attracted to the negatively c... More
Primer
Selecting a Functional Oligonucleotide from a Random Mix
SELEX (Systematic Evolution of Ligands by EXponential enrichment) was first described and used in 1990 to isolate a functional oligonucleotide from a random pool of differently-shaped DNA or RNA molecules.1-2SELEX-derived oligonucleotides (RNA or single-stranded DNA) known as aptamers, bind with hig... More
Primer
Planning Your Next Chemogenetic Experiment
Chemogenetic experiments have been used to study a wide variety of behaviors, such as triggers for substance abuse,eating disorders, obesity, obesity-associated metabolic abnormalities, long term memory, hypertension, locomotor ability, as well as pain, startle, and fear responses. For instance,Hori... More
Primer
Immunodiagnostics in the Post-Covid Era
The benefits of globalization have ushered in the reality of fast-spreading infectious diseases. This has made the quick development of preventive vaccines and immune therapies imperative. Spread of pathogens through rapid and constant travel is compounded by the multipronged challenges ranging from... More
Primer
Dried Blood Spot Testing: Advantages and Disadvantages
In vitro diagnostics, marketed directly to consumers without the involvement of a health care provider are called direct-to-consumer tests, or DTC, which are facilitated through easy-to-collect sample matrices such as dried blood spots (DBS), saliva, or swabs that allow the safe and stable transport... More
Mixer
Organoids for Airway and Alveolar Disease Modeling
In this Mixer, we will discuss the development of respiratory airway and alveolar organoids derived from human iPSCs and how these are used in modeling respiratory diseases such as cystic fibrosis. Airway organoid models can be used for testing the therapeutic efficacy of candidate drugs for respira... More
Mixer
Cell Therapy: Autologous vs Allogeneic Cell Expansion
This Mixer discusses culture systems, integrated consumables, and technical considerations for cell expansion processes in the production of autologous and allogeneic cell therapy products. Our expert panelists will discuss the implementation of scalable, cost-efficient technologies and process opti... More
Mixer
Mapping Tissue Carbohydrates Using Mass Spectrometry
This mixer will be relevant for all interested in methods used in glycoproteomics research that aims to understand the pivotal role of carbohydrate motifs that modify the proteome.Glycoproteomics, a dynamic area of frontier research, provides a systems biology approach to understanding the biologica... More
Mixer
Viral Titer Assays and Interpretations
In this Mixer we will discuss some common methods used to measure viral titers and considerations one should bear in mind when interpreting the results from different assays used to quantify virus particles.Along with all things virology, the recent pandemic has led to focused interest in the accura... More
Mixer
Cell Therapy Development: Tackling Analytic Challenges
Cell therapy products are an exciting new class of medicinal products that offer opportunities to treat and cure diseases that cannot be treated using conventional therapeutic options. The development and manufacturing of cell therapy products pose several challenges compared to other biologics. Yet... More
Video
GEN Protocols Expert Exchanges: Critical ALS Biomarkers of Neuroinflammation & Oxidative Stress
Biomarkers are essential tools for the development of therapeutics, clinical trial design and patient stratification. Biomarkers also help evaluate the efficacy of therapeutic intervention on disease progression or relapse. Yet, biomarker driven patient stratification, a hallmark in oncology trials,... More
Video
GEN Protocols Expert Exchanges: Patient-Derived Pancreatic Cancer Organoids Predict Response to Therapy
Pancreatic cancer accounts forthree percentof all cancers in the United States and is responsible for seven percent of all cancer deaths. Miniature organoids cloned from pancreatic tumors in patients are being grown in laboratories to conduct assays that can predict the effectiveness of chemotherapy... More
Video
GEN Protocols Expert Exchanges: COVID-19 Immunodiagnostic Test Identifies Antibody Source
Need another COVID-19 booster?A new immunodiagnostic test called PictArray helps you decide. The test tells you exactly where your antibodies to the virus are coming from - whether your antibodies are from vaccines or SARS-CoV-2 infections, or both. The test detects SARS-CoV-2 antigens--both spike p... More
Audio
GEN Protocols Expert Exchanges: Next Generation Techniques to Develop Genetic Medicines
The past decade has seen a shift in drug discovery to include targeting the root cause of genetic diseases: loss-of-function, gain-of-function, and change-of-function mutations in the genome. Although first generation genetic medicines like gene therapies heralded a new approach to treating previous... More
Video
GEN Protocols Expert Exchanges: Tools and Techniques to Develop Therapies for Neurological Disorders
The human brain is an exquisitely complex organ, consisting of nearly one hundred billion neurons, an equivalent number of non-neuronal cells and trillions of synapses. Understanding the factors that play a role in the development of the structure and function of the brain is an essential step to de... More
Annual Guide
