The Anti-14-3-3 alpha+beta antibody is widely applied in laboratory protocols designed to identify and study 14-3-3 family proteins, a group of highly conserved regulatory molecules that bind to phosphorylated motifs on target proteins. These proteins play essential roles in signal transduction, protein trafficking, and intracellular localization. In research, the 14-3-3 alpha and beta isoforms are often grouped due to their structural similarity and overlapping functions.
Structural Overview and Classification
The 14-3-3 protein family includes seven human isoforms: beta, gamma, epsilon, eta, sigma, tau, and zeta. Among them, alpha and beta (encoded by YWHAA and YWHAB) are commonly studied together because of near-identical sequences and redundant function in some cellular contexts. These isoforms form homo- and heterodimers, interacting with proteins containing RSXpSXP or RXXXpSXP motifs.
For amino acid sequences and protein structures, consult:
Cellular Distribution and Biological Relevance
14-3-3 alpha and beta are ubiquitously expressed in mammalian tissues, particularly in:
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Neural tissues, where they are involved in synaptic function and axonal transport.
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Skeletal muscle, regulating actin cytoskeleton assembly.
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Hepatic tissues, influencing glycogen synthesis via glycogen synthase kinase-3 (GSK3) binding.
Their distribution in tissue is detailed in the Human Protein Atlas (https://www.proteinatlas.org).
Molecular Interactions and Pathways
These proteins are central in many protein–protein interaction networks. They bind a range of phosphoproteins, including:
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BAD, a Bcl-2 associated agonist of cell regulation.
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Cdc25 phosphatases, which modulate cell progression.
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FOXO transcription factors, affecting cellular aging and oxidative responses.
Molecular interaction maps are available from:
Antibody Production and Host Species
The Anti-14-3-3 alpha+beta antibody is typically raised in rabbit or goat hosts and may be available as polyclonal, monoclonal, or recombinant antibody clones. Purification methods often include protein A/G chromatography or affinity purification using immobilized peptides.
For information on recombinant production:
Applications in Wet-Lab Protocols
1. Western Blotting
In this application, the antibody identifies denatured protein samples separated by SDS-PAGE. Bands corresponding to ~28-30 kDa proteins typically indicate successful detection of 14-3-3 alpha or beta isoforms.
Protocols may reference:
2. Immunoprecipitation (IP)
14-3-3 complexes can be pulled from lysates using this antibody, allowing co-immunoprecipitation of associated signaling proteins like AKT, PKC, and Raf-1.
Refer to:
3. Immunofluorescence (IF) and Immunohistochemistry (IHC)
The antibody is used to visualize intracellular localization in fixed cells or tissues. Co-localization with nuclear markers may indicate transcriptional roles.
4. Enzyme-Linked Immunosorbent Assay (ELISA)
Quantitative detection in serum or lysates using sandwich ELISA format. This method is useful in comparative protein expression analysis under different experimental treatments.
Research Domains and Studies
Protein Stability and Localization
14-3-3 binding often protects targets from degradation. It also influences subcellular trafficking between the nucleus and cytoplasm. In-depth analysis of nuclear export signals is available at:
Signal Cascades
By regulating Raf-MEK-ERK and PI3K-AKT pathways, the 14-3-3 alpha+beta dimer supports cellular response to extracellular signals. More detailed data can be explored via:
Protein-Protein Interaction Assays
Using yeast two-hybrid or mass spectrometry, the binding profile of 14-3-3 isoforms can be mapped. This provides information on network-level regulation.
Explore:
Antibody Validation and Reproducibility
Every batch of antibody must be validated to comply with reproducibility standards. These include:
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Cross-reactivity testing
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Lot-to-lot consistency
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Epitope mapping
The NIH Common Fund and Office of Research Infrastructure Programs outline quality control protocols.
Storage and Handling
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Short-term (≤2 weeks): 4°C with 0.02% sodium azide
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Long-term: -20°C or -80°C in glycerol-containing buffer
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Avoid repeated freeze-thaw cycles
Refer to:
Bioinformatics Resources
Sequence alignment and epitope prediction tools for YWHAB/YWHAA are available via:
Conclusion
The Anti-14-3-3 alpha+beta antibody is a versatile reagent used in many technical workflows to monitor protein expression, binding interactions, and pathway involvement in cells and tissues. As one of the most referenced reagents in cell regulation studies, it is foundational in laboratories focused on intracellular mechanisms. With applications in Western blot, IHC, IP, and ELISA, the antibody contributes to reproducible, high-resolution data generation across experimental platforms. For reproducibility and regulatory compliance, always select well-validated antibody sources and reference standard protocols from .edu and .gov repositories.