Explore AlphaLISA SureFire Ultra formats
AlphaLISA SureFire Ultra
In an AlphaLISA™ SureFire® Ultra™ assay, detection relies on two antibodies: one targeting a phospho-specific site and the other a separate epitope on the same protein. Donor beads are coated with streptavidin to capture the biotinylated antibody while the Acceptor beads are coated with a proprietary CaptSure™ agent that immobilizes the second assay antibody, labeled with a CaptSure™ tag.
AlphaLISA SureFire Ultra Assay Principle
We offer kits to detect "total" analyte content (measuring both phosphorylated and non-phosphorylated forms of the target protein). These total protein assays serve multiple critical functions: they provide essential controls for normalization of data generated with our phosphoprotein-specific kits, enabling researchers to distinguish between true inhibition of phosphorylation versus cell toxicity or modulation of target expression.
These assays are also critical for accelerating Targeted Protein Degradation (TPD) research by comprehensively evaluating the entire TPD workflow-from initial target identification and validation through compound screening and characterization to downstream functional studies-with greater confidence and precision.
AlphaLISA SureFire Biotin Free
AlphaLISA SureFire Biotin Free platform introduces a proprietary "CaptSure 3" coating on the Alpha Donor Bead, replacing the streptavidin coating that has long been associated with legacy Alpha technology.
AlphaLISA SureFire Biotin Free eliminates the need to consider free biotin in cell culture media, such as RPMI 1640, while maintaining high performance.
AlphaLISA SureFire Biotin Free (ASBF) assay principle
AlphaLISA SureFire Ultra Multiplexing
Revvity offers multiplex assays, divided into two categories:
1. Multiplex SureFire Ultra (MPSU) kits are complete kits for phospho and total detection of the same target. Assessing the total protein level (phosphorylated and nonphosphorylated) is essential in distinguishing whether up- or down-regulation of phosphorylation is due to changes in a protein’s expression levels or to cell viability.
Misinterpretation of assay results can potentially lead to higher false positive or false negative rates, or incorrect classification of a compound’s mechanism of action (MoA). The assay principle for MPSU kits is shown schematically below.
Multiplex SureFire Ultra Assay Principle
2. Terbium SureFire Ultra (TBSU) kits measure a single cell signaling target and are intended to be combined with a standard AlphaLISA SureFire Ultra kit (ALSU) to assess the second target. The TBSU kits allow for a mix-and-match combination of any two targets in the assay lists, providing measurement of two separate targets with flexibility. The TBSU kits will report the target of interest through the Terbium (Tb) Acceptor beads (545 nm emission), while the ALSU kit reports via the Europium (Eu) AlphaLISA Acceptor beads (615 nm emission).
Terbium SureFire Ultra Assay Principle
Target area
AlphaLISA SureFire Ultra assays empower researchers to explore a wide range of signaling pathways by enabling precise monitoring of phosphorylation events from membrane receptor activation to nuclear transcriptional responses. The following sections highlight how this technology can be applied across various receptor types and signaling contexts
AlphaLISA SureFire Ultra assays empower researchers to explore a wide range of signaling pathways by enabling precise monitoring of phosphorylation events from membrane receptor activation to nuclear transcriptional responses. The following sections highlight how this technology can be applied across various receptor types and signaling contexts
Signaling pathways
Assessing phosphorylation events in cellular models is particularly crucial during therapeutic drug development, as these post-translational modifications serve as critical nodes in the complex signaling cascades that transmit external stimuli from the cell membrane to the nucleus. By monitoring these phosphorylation events, researchers can trace the complete signal transduction pathway from receptor activation at the cell surface through cytoplasmic kinase cascades to transcriptional regulation in the nucleus; providing valuable insights into a compound's mechanism of action. This comprehensive pathway analysis not only confirms target engagement but also reveals how the drug's effects propagate through interconnected signaling networks, ultimately influencing gene expression and cellular responses. Such detailed pathway deciphering is essential for understanding both on-target efficacy and potential off-target effects that might impact the drug's therapeutic profile.
Example of SYK signaling pathway
GPCR studies
AlphaLISA SureFire Ultra technology enables comprehensive investigation of signal transduction pathways by monitoring the cascade of phosphorylation events that transmit information from cell surface receptors to the nucleus. When G-protein coupled receptors (GPCRs) are activated at the plasma membrane, they trigger distinct signaling cascades depending on their G-protein coupling: Gαi/o-coupled receptors typically activate both the ERK MAP kinase pathway and the PI3K pathway, resulting in phosphorylation of ERK1/2 and Akt proteins respectively, while Gαs- and Gαq-coupled receptors often lead to ERK1/2 and/or CREB phosphorylation, critical transcription factor regulators in the nucleus. By strategically selecting the appropriate AlphaLISA SureFire Ultra kit to monitor these specific phosphorylation events, researchers can trace the complete signal flow from receptor engagement through cytoplasmic kinase networks to nuclear transcriptional regulation. Importantly, these signaling architectures exhibit cell line-specific variations due to differences in receptor expression, pathway component abundance, and regulatory mechanisms, making thoughtful cell model selection crucial for accurately mapping the signal transduction landscape from membrane to nucleus and obtaining physiologically relevant insights into receptor function and drug effects.
Other receptor activation studies
AlphaLISA SureFire Ultra kits provide powerful tools for dissecting the complex signaling networks that connect diverse membrane receptors to nuclear transcriptional responses. Beyond GPCRs, these assays excel at mapping the intricate phosphorylation cascades triggered by receptor tyrosine kinases (RTKs), cytokine receptors, and stress-response pathways, each representing distinct mechanisms by which extracellular signals are transmitted to the nucleus. For instance, when insulin or EGF binds their respective RTKs, they initiate branching signaling cascades involving IRS/PI3K/Akt and Ras/Raf/MEK/ERK pathways that ultimately converge on nuclear transcription factors to regulate metabolism or proliferation. Similarly, inflammatory cytokines like TNFα activate complex signaling networks involving MAP kinases and NFκB pathways that transmit stress signals from the membrane to nuclear transcriptional machinery. By strategically monitoring key phosphorylation events along these pathways, AlphaLISA SureFire Ultra technology enables researchers to trace signal propagation from the initial receptor engagement at the cell surface through cytoplasmic transduction networks to the ultimate nuclear responses, providing comprehensive insights into both normal cellular communication and pathological signaling alterations.
Example of cytokine receptors signaling pathway
Targeted protein degradation
Accelerate Targeted Protein Degradation (TPD) research by monitoring protein expression levels and confidently evaluating the entire TPD workflow-from target identification and validation to compound screening, characterization, and downstream functional studies-with greater precision.
Therapeutic area
These cell-based kinase assays enable precise detection of phosphorylated proteins across a wide range of signaling pathways and therapeutic areas.
These cell-based kinase assays enable precise detection of phosphorylated proteins across a wide range of signaling pathways and therapeutic areas.
Oncology and immuno-oncology: A comprehensive portfolio, including assays for cytokines, immune cells, biomarkers, cell proliferation, cell toxicity and more.
Neuroscience: Investigate intracellular signaling in models of neurodegenerative diseases like Alzheimer’s and Parkinson’s.
Autoimmune and inflammatory diseases: Analyze pathway activation in models of rheumatoid arthritis, lupus, and IBD.
Metabolic disorders: Study insulin signaling and kinase modulation in obesity and diabetes research.
Fibrosis and tissue remodeling: Monitor fibrotic signaling cascades through phospho-target detection.
Virology and infectious disease: Assess host cell signaling responses to viral infection or antiviral compounds.
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