HECW2 Antibody, FITC conjugated
Description
Introduction to HECW2 Antibody, FITC-Conjugated
The HECW2 antibody conjugated with fluorescein isothiocyanate (FITC) is a specialized immunological tool designed for fluorescent detection of the HECW2 protein. HECW2 (HECT, C2, and WW domain-containing E3 ubiquitin protein ligase 2) is an E3 ubiquitin ligase critical for protein ubiquitination, a process regulating protein stability, localization, and degradation. Mutations in HECW2 are associated with neurodevelopmental disorders, including epilepsy, intellectual disability, and autism spectrum features .
Target Protein Overview
HECW2 is a 175.8 kDa cytoplasmic protein involved in ubiquitination-dependent processes. It stabilizes TP73, enhancing transcriptional activation . Mutations in HECW2 disrupt these pathways, leading to neurodevelopmental and epileptic phenotypes .
Use of FITC-Conjugated Antibody
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Immunofluorescence (IF): Enables visualization of HECW2 subcellular localization in fixed cells.
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Flow Cytometry (FC): Detects HECW2 expression in live or fixed cells for quantitative analysis .
Example Workflow:
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Sample Preparation: Fix and permeabilize cells.
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Primary Antibody Incubation: Use HECW2-FITC antibody at recommended dilution.
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Detection: Analyze fluorescence via microscopy or flow cytometry.
HECW2 in Neurodevelopmental Disorders
De novo missense mutations in HECW2 are linked to:
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Developmental Encephalopathy: Early-onset epilepsy, hypotonia, and absent language .
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Autistic Features: Hand-flapping, self-injurious behavior, and stereotypies .
Zebrafish Studies:
Knockdown of hecw2a caused brain morphological abnormalities, supporting its role in neural development .
Diagnostic and Therapeutic Potential
While the FITC-conjugated antibody is not directly cited in clinical studies, HECW2 antibodies (unconjugated) are used in:
Limitations and Considerations
Product Specs
Constituents: 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
- Research indicates that HECW2 interacts with proliferating cell nuclear antigen (PCNA) and lamin B1, both of which bind to lamin A. This interaction is facilitated by a canonical PCNA-interacting protein (PIP) motif within HECW2. HECW2 promotes the ubiquitination of PCNA and lamin B1, ultimately targeting them for degradation via the proteasome. PMID: 29753763
- HECW2, a novel EC ubiquitin E3 ligase, plays a critical role in maintaining the stability of endothelial cell-to-cell junctions by regulating the stability of AMOT-like 1 (AMOTL1). PMID: 27498087
- HECW2 functions as an ubiquitin ligase that stabilizes p73, a critical mediator of neurodevelopment and neurogenesis. This study suggests that pathogenic genetic variations in HECW2 may contribute to neurodevelopmental disorders in humans. PMID: 27389779
- This research provides further support for HECW2 as a potential candidate gene for intellectual disability and epilepsy. In a study of 39 patient-parent trios, 29 de novo mutations in the coding sequence of HECW2 were identified. PMID: 27334371
- Low expression levels of HECW2 have been associated with cervical cancer. PMID: 25156441
- NEDL2, a novel substrate of APC/C-Cdh1, plays a regulatory role in the transition from metaphase to anaphase as cells exit mitosis. PMID: 24163370
Q&A
What experimental applications are validated for FITC-conjugated HECW2 antibodies?
The FITC-conjugated HECW2 antibody (UniProt ID: Q9P2P5) is validated for enzyme-linked immunosorbent assays (ELISA) in human samples . Its polyclonal nature ensures broad epitope recognition, while FITC conjugation enables fluorescence-based detection without secondary antibodies. Researchers should optimize dilutions empirically:
| Application | Recommended Dilution | Buffer Compatibility | Key Validation Metric |
|---|---|---|---|
| ELISA | 1:500–1:5,000 | PBS (pH 7.4) | Linear signal-to-noise ratio across serial dilutions |
How should researchers validate HECW2 antibody specificity in novel experimental systems?
Specificity validation requires three parallel approaches:
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Knockdown/knockout controls: Compare signal intensity in HECW2-depleted cells (e.g., siRNA-treated) versus wild-type .
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Immunoblotting: Confirm a single band at ~250 kDa (predicted molecular weight of HECW2) in Western blots .
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Blocking peptide assays: Pre-incubate the antibody with recombinant HECW2 (495–641AA) to observe signal reduction .
The antibody’s immunogen (residues 495–641) contains the HECT domain critical for ubiquitin ligase activity, making it suitable for functional studies .
What are the storage and stability considerations for FITC-conjugated antibodies?
Store aliquots at -20°C or -80°C in 50% glycerol/PBS to prevent freeze-thaw degradation . Post-thaw, centrifuge at 12,000×g for 5 minutes to remove aggregates. FITC fluorescence remains stable for 6–12 months under these conditions, but researchers should:
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Avoid prolonged light exposure (FITC photobleaching half-life: ~2 hours under standard microscopy conditions).
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Verify performance monthly using a positive control (e.g., HEK293T cells overexpressing HECW2) .
How can this antibody resolve contradictory data in HECW2 subcellular localization studies?
Conflicting reports on HECW2 localization (nuclear vs. cytoplasmic) often arise from fixation methods or epitope accessibility. To address this:
Protocol for Dual-Labeling Immunofluorescence
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Fixation: Use 2% formaldehyde (20 min) followed by 0.1% Triton X-100 permeabilization .
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Antibody cocktail: Combine FITC-conjugated HECW2 (1:200) with organelle markers (e.g., Lamin B1 for nucleus).
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Image quantification: Calculate Manders’ overlap coefficient (MOC) across ≥50 cells .
In porcine oocytes, this approach revealed HECW2’s dual localization: nuclear during zygote formation and cytoplasmic in mature gametes .
What mechanistic insights can be gained by combining this antibody with ubiquitination assays?
HECW2’s role as an E3 ligase for TP73 stabilization can be profiled via:
Co-Immunoprecipitation (Co-IP) Workflow
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Transfect cells with HA-tagged ubiquitin and HECW2.
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Lyse cells in RIPA buffer + 10 μM MG132 (proteasome inhibitor).
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Immunoprecipitate TP73 using magnetic beads.
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Detect ubiquitinated TP73 via Western blot and HECW2-ubiquitin complexes via FITC fluorescence .
Key finding: HECW2-mediated ubiquitination increases TP73α transcriptional activity by 3.2-fold in HEK293 cells (p < 0.01) .
How does HECW2 antibody performance vary across disease models, such as atherosclerosis?
In ox-LDL-induced endothelial injury models:
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Circ_HECW2/miR-942-5p axis: FITC-conjugated antibodies quantified HECW2 upregulation (2.1-fold vs. controls, p < 0.001) via flow cytometry .
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Functional validation: siRNA knockdown reduced apoptotic hCMECs by 38% (Annexin V/PI assay), confirming target engagement .
| Parameter | ox-LDL Model | siRNA-Treated | p-value |
|---|---|---|---|
| HECW2 Fluorescence | 215 ± 12 AU | 89 ± 8 AU | <0.001 |
| Apoptotic Cells (%) | 27.3 ± 2.1 | 16.8 ± 1.4 | <0.01 |
What are the limitations of using this antibody in cross-species studies?
While raised against human HECW2, the antibody shows limited reactivity in non-primate models:
| Species | Reactivity | Recommended Validation Step |
|---|---|---|
| Porcine | Moderate | Pre-adsorption with porcine liver extract |
| Murine | None | Use alternative clones (e.g., Mouse Mono 1A3) |
In porcine zygotes, 50 μg/mL antibody injection reduced fertilization rates by 22% (p < 0.05), suggesting off-target effects in non-human systems .
How can researchers optimize this antibody for multiplexed imaging workflows?
Combine with TRITC-conjugated secondary antibodies using:
