HECW2 Antibody, Biotin conjugated

What is HECW2 and why is it important in research?

HECW2 (HECT, C2 and WW domain containing E3 ubiquitin protein ligase 2) is a member of the Nedd4 family of HECT domain E3 ubiquitin ligases that plays crucial roles in neurodevelopment and neurogenesis. It functions primarily by stabilizing p73, a member of the p53 family with specific neurodevelopmental expression patterns . Research interest in HECW2 has increased significantly since the discovery that pathogenic variants in the HECW2 gene are associated with neurodevelopmental disorders characterized by intellectual disability, hypotonia, seizures, and vision problems . The protein is involved in the ubiquitination process, which regulates protein degradation and cellular signaling, making it an important target for understanding both normal neurodevelopment and pathological conditions.

What are the key specifications of HECW2 antibody with biotin conjugation?

The HECW2 rabbit polyclonal antibody with biotin conjugation is specifically designed for detecting the HECW2 protein in human and mouse samples . This primary antibody has the following characteristics:

• Host: Rabbit

• Clonality: Polyclonal

• Isotype: IgG

• Conjugation: Biotin

• Reactivity: Human and Mouse

• Epitope: Internal region of HECW2

• Recommended dilutions: ELISA 1:1000; Western Blot 1:100-500

The antibody targets the internal region of HECW2, allowing for specific detection of this protein in various experimental contexts .

What detection techniques can utilize biotin-conjugated HECW2 antibodies?

Biotin-conjugated HECW2 antibodies offer versatility in detection methodologies due to the strong affinity between biotin and streptavidin. The most common applications include:

• ELISA assays: The biotin conjugation enhances detection sensitivity when used at a recommended dilution of 1:1000 .

• Western blot analysis: At dilutions of 1:100-500, these antibodies provide specific detection of HECW2 protein in cell or tissue lysates .

• Immunohistochemistry: Though not explicitly mentioned in the search results, biotin-conjugated antibodies generally work well in IHC applications.

• Protein detection using streptavidin-based systems: Biotinylated proteins can be detected using HRP-conjugated streptavidin in detection workflows .

The biotin-streptavidin interaction provides an amplification step that increases detection sensitivity compared to direct detection methods.

How should experimental controls be designed when using HECW2 biotin-conjugated antibodies?

When designing experiments with HECW2 biotin-conjugated antibodies, researchers should implement the following control strategies:

• Negative controls:

  • Samples known to lack HECW2 expression

  • Isotype controls using non-specific rabbit IgG with biotin conjugation

  • Secondary-only controls (streptavidin-detection system without primary antibody)

• Positive controls:

  • Cell lines or tissues known to express HECW2

  • Recombinant HECW2 protein (particularly the region containing amino acids 495-641, which is used as an immunogen in some commercially available antibodies)

• Validation controls:

  • Knockdown or knockout models for HECW2 to confirm specificity

  • Competitive blocking with recombinant HECW2 protein

These controls help distinguish true signals from background and validate the specificity of the antibody in your experimental system.

What is the optimal sample preparation protocol for HECW2 detection in neuronal tissues?

For neuronal tissues, which are particularly relevant given HECW2's role in neurodevelopment, the following sample preparation protocol is recommended:

• Tissue fixation and processing:

  • For fresh tissues: Use 4% paraformaldehyde fixation followed by cryoprotection in sucrose gradients

  • For paraffin embedding: Ensure antigen retrieval steps are optimized (typically heat-mediated retrieval in citrate buffer pH 6.0)

• Lysate preparation for Western blot:

  • Homogenize tissue in RIPA buffer supplemented with protease inhibitors

  • Include phosphatase inhibitors if phosphorylated forms are of interest

  • Gentle sonication may help release nuclear proteins

  • Centrifuge at 14,000g for 15 minutes at 4°C to clear debris

• Blocking endogenous biotin:

  • Critical for brain tissues which contain high levels of endogenous biotin

  • Pre-block with avidin-biotin blocking kit before antibody incubation

• Buffer considerations:

  • Store antibody in 50% glycerol with 0.01M PBS (pH 7.4) and 0.03% Proclin 300 as preservative

  • For dilutions, use PBS with 1-5% BSA or normal serum from the species of the secondary reagent

Proper sample preparation is essential for maintaining protein integrity while ensuring accessibility of the epitope for antibody binding.

How can researchers optimize detection sensitivity when using biotin-conjugated HECW2 antibodies?

To maximize detection sensitivity with biotin-conjugated HECW2 antibodies:

• Signal amplification techniques:

  • Implement tyramide signal amplification (TSA) systems compatible with biotin-streptavidin interactions

  • Use high-sensitivity HRP-conjugated streptavidin or neutravidin for detection

  • Consider multi-layer approaches (biotin-streptavidin-biotin) for further amplification

• Dilution optimization:

  • Perform titration experiments to determine optimal antibody concentration (starting with recommended 1:1000 for ELISA and 1:100-500 for Western blot)

  • Balance signal strength with background minimization

• Incubation parameters:

  • Extend primary antibody incubation time (overnight at 4°C rather than 1-2 hours at room temperature)

  • Optimize temperature conditions for antigen-antibody binding

• Detection substrate selection:

  • Use enhanced chemiluminescence (ECL) substrates with extended signal duration

  • For fluorescent applications, select streptavidin conjugates with bright, photostable fluorophores

• Background reduction:

  • Include 0.1-0.3% Triton X-100 in blocking solutions to reduce non-specific binding

  • Use casein-based blockers instead of BSA when background is problematic

These approaches can significantly improve signal-to-noise ratio when detecting low-abundance HECW2 protein in experimental samples.

How can HECW2 antibodies contribute to understanding neurodevelopmental disorders?

The discovery of HECW2 variants in patients with neurodevelopmental disorders provides an important research avenue where HECW2 antibodies can make significant contributions:

• Genotype-phenotype correlation studies:

  • Detecting altered HECW2 protein levels or localization in patient-derived samples

  • Comparing HECW2 expression patterns between patients with different variants (particularly the recurrent variants p.(Arg1191Gln), p.(Asn1199Lys), p.(Phe1327Ser), and p.(Arg1330Trp))

• Functional consequence assessment:

  • Evaluating how HECW2 variants affect its ubiquitin ligase activity

  • Measuring changes in p73 stability, a known HECW2 substrate critical for neurodevelopment

  • Investigating alterations in subcellular localization of mutant HECW2 versus wild-type

• Developmental expression mapping:

  • Comparing HECW2 expression patterns across brain regions during development

  • Co-localization studies with neuronal and glial markers

  • Temporal expression analysis during critical neurodevelopmental windows

• Therapeutic target identification:

  • Screening for compounds that may stabilize HECW2 function or compensate for its dysfunction

  • Monitoring HECW2 levels in response to potential treatment strategies

The biotin conjugation provides flexibility in detection systems, enabling multicolor imaging alongside other markers of neuronal development or function .

What techniques can elucidate the functional relationship between HECW2 and p73 in neural development?

To investigate the critical HECW2-p73 axis in neural development:

• Co-immunoprecipitation studies:

  • Use biotin-conjugated HECW2 antibodies to pull down HECW2 complexes

  • Detect p73 in the precipitated material to confirm interaction

  • Compare wild-type versus variant HECW2 proteins for changes in p73 binding efficiency

• Ubiquitination assays:

  • Detect ubiquitinated p73 species in the presence of normal or mutant HECW2

  • Quantify differences in p73 stability and half-life under various HECW2 conditions

  • Investigate how HECW2 variants found in neurodevelopmental disorders affect p73 ubiquitination patterns

• Transcriptional activity measurement:

  • Use reporter assays to measure p73 transcriptional activity as influenced by HECW2

  • Compare transcriptional outputs between wild-type and mutant HECW2 conditions

  • Identify p73 target genes whose expression is differentially affected by HECW2 variants

• Neural differentiation models:

  • Monitor HECW2-p73 interactions during neural stem cell differentiation

  • Assess how disruption of this interaction affects neurogenesis and neuronal maturation

  • Create time-course analyses of HECW2 and p73 levels during critical developmental stages

These approaches can reveal mechanistic insights into how HECW2 mutations lead to neurodevelopmental phenotypes through disruption of p73-dependent processes .

What are the optimal approaches for studying HECW2 in induced pluripotent stem cell (iPSC)-derived neurons?

For investigating HECW2 in iPSC-derived neuronal models, the following approaches are recommended:

• Neural differentiation protocol optimization:

  • Monitor HECW2 expression throughout differentiation from iPSCs to neural progenitors to mature neurons

  • Compare differentiation efficiency between control and HECW2-variant containing lines

  • Establish timeline of HECW2 expression relative to neural markers

• Immunostaining protocols:

  • Use biotin-conjugated HECW2 antibodies at 1:100-200 dilution in neuronal culture immunocytochemistry

  • Perform co-staining with neuronal markers (MAP2, TUJ1) and p73

  • Include analysis of subcellular localization in developing neurons

• Functional assessments:

  • Electrophysiological characterization of neurons with normal versus altered HECW2 function

  • Calcium imaging to assess neuronal activity patterns

  • Synaptic marker analysis to evaluate synaptogenesis

• Single-cell approaches:

  • Combine with scRNA-seq to correlate HECW2 protein levels with transcriptional profiles

  • Use spatial transcriptomics alongside immunostaining to map HECW2 function in developing neuronal networks

• CRISPR-engineered isogenic lines:

  • Generate iPSC lines with specific HECW2 variants matching those found in patients

  • Create reporter lines to monitor HECW2 activity in real-time during differentiation

These methodologies are particularly valuable for modeling human neurodevelopmental disorders associated with HECW2 variants in a physiologically relevant cellular context.

How can researchers address non-specific binding when using biotin-conjugated HECW2 antibodies?

Non-specific binding is a common challenge when working with biotin-conjugated antibodies. Here are specific approaches to minimize this issue:

• Endogenous biotin blocking:

  • Use commercial avidin/biotin blocking kits before antibody application

  • For tissues with high endogenous biotin (brain, kidney, liver), extend blocking times

  • Consider alternative detection systems if endogenous biotin remains problematic

• Optimize blocking conditions:

  • Test different blocking agents (BSA, normal serum, commercial blockers)

  • Increase blocking time (from 1 hour to overnight at 4°C)

  • Add 0.1-0.3% detergent (Triton X-100 or Tween-20) to reduce hydrophobic interactions

• Antibody dilution optimization:

  • Perform titration experiments to find the optimal concentration that maximizes specific signal while minimizing background

  • Start with the recommended dilutions (1:1000 for ELISA; 1:100-500 for Western blot)

• Pre-adsorption control:

  • Pre-incubate the antibody with recombinant HECW2 protein

  • Compare results with and without pre-adsorption to identify non-specific signals

• Secondary detection system considerations:

  • Use highly purified streptavidin conjugates

  • Consider streptavidin alternatives like NeutrAvidin that have lower non-specific binding

Careful optimization of these parameters can significantly improve signal specificity when working with biotin-conjugated HECW2 antibodies.

What are the best practices for storage and handling to maintain antibody integrity?

To preserve the functionality of biotin-conjugated HECW2 antibodies:

• Storage conditions:

  • Store concentrated antibody at -20°C or -80°C to prevent degradation

  • Avoid repeated freeze-thaw cycles by preparing single-use aliquots

  • Use a storage buffer containing 50% glycerol, 0.01M PBS (pH 7.4), and 0.03% Proclin 300 as preservative

• Working solution handling:

  • Prepare fresh dilutions for each experiment

  • Keep working solutions on ice during experiment preparation

  • Return concentrated stock to -20°C immediately after use

• Contamination prevention:

  • Use sterile techniques when handling antibody solutions

  • Filter buffers to remove particulates that might cause aggregation

  • Use clean, DNase/RNase-free tubes for storage

• Transportation considerations:

  • Ship on dry ice for long distances

  • Use insulated containers with cold packs for short-term transportation

• Stability monitoring:

  • Include positive controls in each experiment to track antibody performance over time

  • Document lot numbers and performance characteristics

  • Consider preparing a standard curve with recombinant protein for quantitative applications

Proper storage and handling are essential for maintaining antibody specificity and sensitivity throughout the research project timeline.

How can researchers validate antibody specificity for HECW2 detection in new experimental models?

When adapting HECW2 antibodies to new experimental systems, validation is crucial:

• Genetic validation approaches:

  • Compare staining patterns in HECW2 knockout/knockdown versus wild-type samples

  • Use CRISPR-Cas9 to generate HECW2-deficient cell lines as negative controls

  • Perform rescue experiments by reintroducing HECW2 to knockout models

• Orthogonal detection methods:

  • Confirm protein expression using alternative antibodies targeting different HECW2 epitopes

  • Correlate protein detection with mRNA expression (RT-qPCR or RNA-seq)

  • Use mass spectrometry to confirm antibody-detected bands contain HECW2 peptides

• Cross-species validation:

  • For new animal models, confirm sequence homology in the epitope region

  • Test antibody performance in species with known vs. unknown reactivity

  • Consider species-specific positive controls when working with non-human/mouse samples

• Antigen competition:

  • Pre-incubate antibody with recombinant HECW2 protein (particularly the 495-641AA region)

  • Verify signal reduction in competition experiments

  • Include concentration gradients of competing antigen

• Multi-technique confirmation:

  • Verify HECW2 detection across multiple techniques (Western blot, ICC, IHC, IP)

  • Compare subcellular localization patterns with published literature

  • Confirm molecular weight of detected protein matches predicted HECW2 size

Thorough validation not only ensures experimental reliability but also contributes to addressing the reproducibility challenges in antibody-based research.

How might HECW2 antibodies facilitate investigation of genotype-phenotype correlations in neurodevelopmental disorders?

Biotin-conjugated HECW2 antibodies provide valuable tools for exploring the relationship between specific genetic variants and clinical phenotypes:

• Variant-specific protein behavior:

  • Compare subcellular localization of wild-type versus mutant HECW2 proteins

  • Assess whether specific variants (particularly recurrent mutations like p.(Arg1191Gln) and p.(Arg1330Trp)) show consistent protein expression patterns

  • Determine if HECT domain variants correlate with different protein interactions compared to variants in other domains

• Domain-specific function analysis:

  • Investigate whether variants in or near the HECT domain (where 88.2% of pathogenic variants are located) show distinct functional consequences

  • Compare how mutations in different protein domains affect HECW2's interaction with p73

  • Correlate protein domain alterations with specific aspects of the clinical phenotype

• Patient-derived models:

  • Analyze HECW2 expression and localization in patient-derived cells

  • Compare patient samples with specific variants to the clinical phenotypes (seizures, visual impairment, gastrointestinal issues)

  • Create isogenic lines with single variants to isolate their effects

• Biomarker development:

  • Assess whether specific alterations in HECW2 detection patterns could serve as diagnostic biomarkers

  • Correlate protein expression patterns with disease severity measures

  • Examine whether HECW2 alterations precede clinical manifestations

These approaches can help explain why certain HECW2 variants are associated with specific clinical features, such as why HECT domain variants more frequently correlate with cortical visual impairment and gastrointestinal issues .

What novel methodologies could enhance the study of HECW2's role in the ubiquitin-proteasome system?

Advanced approaches for investigating HECW2's E3 ligase function include:

• Proximity-dependent labeling:

  • BioID or TurboID fusion with HECW2 to identify proximal interacting proteins

  • APEX2 proximity labeling to map the HECW2 interaction network in living cells

  • Compare wild-type and disease-associated variant interactomes

• Live-cell ubiquitination monitoring:

  • FRET-based sensors to detect HECW2-mediated ubiquitination events in real-time

  • Fluorescently-tagged ubiquitin to track dynamic changes in HECW2 substrates

  • Correlation of ubiquitination patterns with neurodevelopmental processes

• Targeted degradation approaches:

  • PROTACs (Proteolysis Targeting Chimeras) to modulate HECW2 levels

  • Inducible degron systems to study temporal requirements for HECW2

  • Assessment of compensatory mechanisms after acute HECW2 depletion

• Structural biology integration:

  • Combining antibody epitope mapping with structural studies of HECW2

  • Using antibodies to stabilize HECW2 conformations for cryo-EM analysis

  • Structure-guided design of tools to modulate specific HECW2 functions

• Single-molecule approaches:

  • Direct visualization of individual ubiquitination events mediated by HECW2

  • Measuring kinetics of HECW2-substrate interactions at the single-molecule level

  • Correlating molecular behavior with cellular phenotypes

These methodologies could provide unprecedented insights into how HECW2 selects and modifies its substrates, particularly p73 and the APC/C-Cdh1 complex, which are critical for neurodevelopment .

How can multiplexed imaging approaches with HECW2 antibodies advance neurodevelopmental research?

Multiplexed imaging strategies offer powerful approaches for contextualizing HECW2 function:

• Multiparameter imaging combinations:

  • Utilize biotin-conjugated HECW2 antibodies alongside fluorophore-conjugated antibodies against developmental markers

  • Combine with cell-type specific markers to map HECW2 expression across neural cell populations

  • Co-stain with subcellular markers to precisely localize HECW2 within cellular compartments

• Cyclic immunofluorescence approaches:

  • Sequential imaging with antibody stripping and reprobing

  • Integration of HECW2 detection in cycles with other proteins of interest

  • Building comprehensive protein interaction maps in the context of tissue architecture

• Spatial transcriptomics integration:

  • Correlate HECW2 protein localization with transcriptional profiles in the same tissue section

  • Map the relationship between HECW2 protein levels and expression of p73-regulated genes

  • Identify regional variations in HECW2 activity across brain structures

• Temporal imaging in developmental models:

  • Live imaging in zebrafish or other transparent model organisms

  • Time-lapse studies of HECW2 dynamics during critical developmental windows

  • Correlation of HECW2 localization changes with morphological development

• Super-resolution approaches:

  • STORM/PALM imaging to visualize nanoscale distribution of HECW2

  • Expansion microscopy to physically enlarge samples for enhanced resolution

  • Correlative light and electron microscopy to connect HECW2 localization with ultrastructural features

These multiplexed approaches can reveal how HECW2 functions within the complex cellular environment of the developing nervous system, potentially explaining the connection between molecular dysfunction and clinical manifestations in patients with HECW2 variants .