Rae1 Antibody
Description
Rae1 Antibody: Definition and Applications
Rae1 antibodies are immunological reagents designed to detect and quantify Rae1 protein in various experimental settings. These antibodies enable researchers to investigate Rae1’s localization, expression levels, and interactions in normal and pathological conditions.
Key Applications:
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Western Blot (WB): Detects Rae1 in lysates (e.g., BxPC-3, HEK-293, HepG2 cells) at dilutions of 1:500–1:2000 .
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Immunohistochemistry (IHC): Identifies Rae1 in formalin-fixed tissues (e.g., human testis) with antigen retrieval .
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Immunofluorescence (IF): Localizes Rae1 in cytoplasmic and nuclear compartments, particularly in tumor cells .
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Flow Cytometry: Quantifies Rae1 surface expression on immune cells (e.g., RAW 264.7 monocytes) using monoclonal antibodies like MAB17582 .
Role in Cancer and Mitosis
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Tumor Growth: Rae1 is overexpressed in colorectal cancer (CRC) and promotes tumor growth by stabilizing spindle bipolarity, inhibiting apoptosis, and inducing chemoresistance .
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Mitotic Checkpoint: Rae1 haplo-insufficiency disrupts mitotic checkpoints, leading to chromosome missegregation and embryonic lethality in mice .
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Hippo Pathway Interaction: Rae1 enhances Hippo signaling by stabilizing cyclin B levels, linking mitotic regulation to organ size control .
Immune Regulation
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Rae1 serves as a ligand for NKG2D, an activating receptor on natural killer (NK) cells. Its expression on stressed or tumor cells triggers immune surveillance .
Validation and Technical Considerations
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Specificity: Monoclonal antibodies (e.g., clones 52A, 60A) show high sensitivity in flow cytometry and ELISA, with minimal cross-reactivity .
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Staining Protocols: For IHC, citrate buffer (pH 6.0) or TE buffer (pH 9.0) is recommended for antigen retrieval .
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Storage: Lyophilized antibodies remain stable for 12 months at -20°C to -70°C; reconstituted aliquots should avoid freeze-thaw cycles .
Emerging Therapeutic Potential
Q&A
Here’s a structured collection of FAQs tailored to academic research scenarios involving Rae1 antibodies, incorporating methodological insights and data from peer-reviewed sources:
What experimental applications are validated for Rae1 antibodies in human cell models?
Rae1 antibodies are widely used for:
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Co-immunoprecipitation (Co-IP): Anti-Rae1 antibodies co-precipitate NuMA and dynein in HeLa cells, confirming protein-protein interactions critical for spindle assembly .
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Immunofluorescence (IF): Methanol-fixed HeLa cells stained with affinity-purified anti-Rae1 antibodies show nuclear/cytoplasmic localization .
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Western blot (WB): Recombinant anti-Rae1 antibodies (e.g., ab124783) detect a 41 kDa band in lysates from MCF7, HeLa, and HEK-293 cells .
Key validation data:
| Application | Cell Line | Antibody Clone | Result | Source |
|---|---|---|---|---|
| WB | HeLa | EPR6923 | 41 kDa | |
| IF | HeLa | Custom polyclonal | Nuclear/cytoplasmic signal |
How do researchers validate Rae1 antibody specificity?
Methodological steps include:
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Knockdown/knockout controls: Use siRNA or CRISPR to reduce Rae1 expression and confirm loss of signal .
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Peptide blocking: Pre-incubate antibodies with immunizing peptides (e.g., residues 313–327 of human Rae1) to assess signal reduction .
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Cross-reactivity testing: Validate species specificity (e.g., ab124783 reacts with human but not mouse/rat ).
Example: A study using HA-tagged Rae1 in keratinocytes confirmed antibody failure in ChIP-seq, prompting epitope tagging as an alternative .
How can Rae1 antibodies resolve contradictions in subcellular localization studies?
Discrepancies (e.g., nuclear vs. cytoplasmic localization) arise from:
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Fixation methods: Methanol fixation preserves Rae1-NuMA interactions, while paraformaldehyde may mask epitopes .
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Antibody clonality: Polyclonal vs. monoclonal antibodies (e.g., ab124783 vs. MAB17582) target distinct epitopes .
Resolution strategy:
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Combine IF with subcellular fractionation and WB.
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Compare multiple clones (e.g., monoclonal EPR6923 vs. polyclonal 20491-1-AP) .
What advanced techniques require carrier-free Rae1 antibodies?
Carrier-free formulations (e.g., ab248002) are critical for:
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Multiplex imaging: Conjugation with metal isotopes (CyTOF) or fluorophores without interference .
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In vivo functional studies: Reduced background in mouse tumor models using BSA/azide-free formats .
Protocol note: For mass cytometry, pair ab248002 with Fluidigm’s Maxpar® labeling kit .
How do Rae1 antibodies enable mechanistic studies of NuMA/HDAC interactions?
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Chromatin binding assays: HA-tagged Rae1 rescues ChIP-seq failures caused by low-affinity commercial antibodies .
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Co-IP workflows: Use RIPA buffer + protease inhibitors to co-precipitate Rae1-NuMA-HDAC complexes .
Data contradiction example: NuMA co-precipitates with Rae1 but not Nup98, suggesting context-dependent complexes .
