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GEN Protocols

OMICS

OMICS

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

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

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

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

Investigating the increased lifespan in C. elegans daf-2 mutants by 4D-Lipidomics™

The small nematode Caenorhabditis elegans is one of the premier biomedical model organisms and employed in many aspects of basic and applied science.... More


Protocol

IsoCode Single-Cell Adaptive Immune: Human Bispecific Protocol

Overview of ProtocolDay 1: Cryopreserved T cells are thawed and cultured overnight in the presence of IL-2.Day 2: Enrichment, Staining and Antigen Stimulation of CD4+ and/or CD8+ T cells for 36-48 hours.Day3: Loading of T cells onto IsoCode chip.... More


Protocol

Single-Cell Intracellular Proteome: HeLa Cell Protocol

Overview of Protocol:Day 1-3: Cryopreserved HeLa cells are thawed and cultured for 72 hours.Day 4: Staining and stimulating of HeLa cells followed by immediate loading onto IsoCode Chip... More


Protocol

IsoCode Single-Cell Adaptive Immune: TCR-T Protocol

A. Overview of ProtocolDay 1: Cryopreserved TCR-T cells are thawed and cultured overnight in the presence of IL-2. Cryopreserved target cell lines are thawed, cultured and passaged several days prior to beginning the experiment.Day 2: Enrichment, Staining, and Antigen Stimulation of CD4+ and/or CD8+... More


Protocol

IsoCode Single-Cell Innate Immune: Human Monocyte Protocol Using Membrane Stain

A. OverviewOverview of ProtocolDay 1: Cryopreserved cells are thawed and cultured overnight in complete RPMI media.Day 2: Enrichment and Stimulation of Monocytes for 24 hours.Day 3: Staining and Loading of Monocytes onto IsoCode Chip.... More


Protocol

IsoCode Single-Cell Innate Immune: Human CD34+ Myeloid Stem Cell Protocol

A. Overview1. Overview of ProtocolDay 1: Cryopreserved cells are thawed and cultured overnight in the presence of IL-2.Day 2: Enrichment and Stimulation of CD34+ cells for 24 hours.Day 3: Staining and Loading of CD34+ cells onto IsoCode chip.NOTE: This protocol outlines the standard method for thawi... More


Protocol

IsoCode Single-Cell Adaptive Immune: TCR-T Protocol

A. Overview of ProtocolDay 1: Cryopreserved TCR-T cells are thawed and cultured overnight in the presence of IL-2. Cryopreserved target cell lines are thawed, cultured and passaged several days prior to beginning the experiment.Day 2: Enrichment, Staining, and Antigen Stimulation of CD4+ and/or CD8+... More


Protocol

IsoCode Single-Cell Innate Immune: Human Monocyte Protocol Using Membrane Stain

A. OverviewOverview of ProtocolDay 1: Cryopreserved cells are thawed and cultured overnight in complete RPMI media.Day 2: Enrichment and Stimulation of Monocytes for 24 hours.Day 3: Staining and Loading of Monocytes onto IsoCode Chip.... More


Protocol

IsoCode Single-Cell Innate Immune: Human CD34+ Myeloid Stem Cell Protocol

A. Overview1. Overview of ProtocolDay 1: Cryopreserved cells are thawed and cultured overnight in the presence of IL-2.Day 2: Enrichment and Stimulation of CD34+ cells for 24 hours.Day 3: Staining and Loading of CD34+ cells onto IsoCode chip.NOTE: This protocol outlines the standard method for thawi... More


Protocol

Multiplexed, trackable CRISPR-based genome engineering for optimization of heterologous protein expression and activity in E. coli

This work demonstrates the use of the Onyx platform for interrogation and optimization of heterologous proteins in E. coli. Compared to traditional methods for protein engineering, such as random mutagenesis or targeted site-directed mutagenesis approaches, the Onyx technology dramatically increases... More


Protocol

4D-Lipidomics™ workflow for increased throughput

Lipid profiling from complex lipid extracts can be a challenging and time consuming task. The high complexity of samples and co-elution of isobaric or isomeric compounds complicate the confident annotation of lipids. The presented 4D-Lipidomics workflow simplifies and streamlines the annotation and ... More


Protocol

Rapid metabolic phenotyping in SARS-CoV-2 patients using the impact II QTOF MS system

The impact II QTOF system meets the critical elements of sensitivity, selectivity, accuracy, and precision for targeted metabolite analysis. The continuous collection of both MS and MS/MS data provides an exponentially deeper and broader pool of data valuable for subsequent retrospective discovery o... More


Protocol

Sorting of water-in-oil emulsions with bacteria using a fluorescent nucleic acid probe

To isolate water-in-oil emulsion droplets containing bacteria from those that do not contain bacteriawe have developed a simple and reliable selective sorting method that allows bacteria-containing droplets to be isolated on our microfluidic chip cell sorter, On-chip Sort. Bacteria-containing drople... More


Protocol

Filter Capture, Extraction, and Sequencing of Coastal Metagenomes

In order to analyze the metagenomes of coastal microbial communities, we have standardized a protocol to collect, extract, and sequence near-shore microbiome samples. This procedure involves on-site filter capture of microbiomes from coastal water, DNA extraction via ceramic bead processing and sili... More


Protocol

Detergent-Free Nuclei Isolation

A novel spin column-based approach to isolate intact single nuclei from tissues (fresh or frozen) and cells without using a detergent. The protocol is fast, easy, and reliable, producing high-quality single nuclei with minimized clumping and possible leakage of the nuclear envelope. The protocol 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


Application

Access the Breadth of Sample Preparation Protocols to Awaken True Functional Immune Biology

Precedent Protocols and Processes ensure that enrichment, stimulation and labeling will result in maximizing the correlative data achieved, while minimizing non-specific impact on the cells themselves. Highlights: • Published enrichment, stimulation, and staining protocols throughout immunotherapy ... More


Primer

Source and Elimination of Common Mass Spectrometry Contaminants

Sensitivity is a key attribute of mass spectrometers to allow quantification of minute amounts of analytes. Yet this sensitivity can be a conundrum. Myriad sources can contribute contaminants that can impact background and confound interpretation of results. An increased background signal can make i... 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

Single-Cell RNA Sequencing Upgrades Sensitivity

A new method for DNA amplification and sequencing called TAS-Seq improves the accuracy of single-cell RNA sequencing (scRNA-seq). TAS-Seq uses a terminal transferase enzyme that amplifies and sequences DNA complementary to mRNA (cDNA) in solid phase on a nanowell/magnetic bead-based cell isolation p... More


Primer

Cyclic Microchip Assay Enhances Throughput of Protein Detection in Single Cells

Most existing single-cell protein analysis methods can analyze about a dozen proteins at a time when the execution of nearly every conceivable cellular function depends on a complex and diverse proteome.A new microchip assay called CycMIST1 (single-cell cyclic multiplex in situ tagging) can measure ... More


Primer

Solvent Precipitation Method Increases Protein Recovery for Proteomics

Study designs in proteomics aim for inclusivity—to characterize every protein in a sample. However, extracting proteins from complex biological samples involves the use of harsh buffers containing detergents and salts that are incompatible with the proteomics workflow. More proteins are lost in atte... 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

Spatial Biology Tools and Techniques

Single-cell spatial phenotyping is transforming biomedical research in fields ranging from oncology to neuroscience. This GEN Protocols Mixer focuses on technological developments and protocols that have enabled spatial approaches in biology. We will explore novel applications of the technology in d... More


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