HECW2 Antibody, HRP conjugated

What is HECW2 and why is it significant for research?

HECW2 (HECT, C2 and WW Domain Containing E3 Ubiquitin Protein Ligase 2) is a HECT-type ubiquitin ligase that mediates the ubiquitination of target proteins including TP73, thereby regulating their stability and function . The significance of HECW2 in research spans multiple fields including cancer biology and neurodevelopment. Recent studies have identified HECW2 as a potential contributor to colorectal cancer progression and chemoresistance through its ability to activate the AKT/mTOR signaling pathway . Additionally, mutations in HECW2 have been associated with intellectual disability and epilepsy, suggesting its crucial role in neuronal development and function . Understanding HECW2's functions requires specialized antibodies that can reliably detect the protein in various experimental contexts.

What are the key applications for HECW2 antibody, HRP conjugated?

HECW2 antibody, HRP conjugated is primarily optimized for enzyme-linked immunosorbent assay (ELISA) applications, where the direct horseradish peroxidase conjugation eliminates the need for secondary antibodies . This provides a significant advantage in terms of procedural efficiency and reduction of non-specific background signals. While ELISA represents the primary application, some HRP-conjugated HECW2 antibodies may also be suitable for Western blotting with appropriate protocol optimization . The recommended dilution for ELISA applications is typically 1:1000, while Western blot applications may require a higher concentration (1:100-500) . When selecting an HRP-conjugated HECW2 antibody, researchers should confirm the validation status for their specific application of interest.

What epitopes of HECW2 are targeted by commercially available antibodies?

Various epitopes of HECW2 are targeted by different commercially available antibodies, allowing researchers to select the most appropriate one for their specific research question. The following table summarizes key epitope regions and their corresponding applications:

The choice of epitope can significantly impact experimental outcomes, particularly when studying HECW2 domains with specific functions . For instance, targeting the HECT domain (which contains the catalytic site for ubiquitin transfer) might be particularly relevant when investigating HECW2's enzymatic activity, while antibodies targeting the C2 or WW domains might be more appropriate for studies focused on protein-protein interactions or membrane localization.

How should HECW2 antibody, HRP conjugated be stored and handled?

Proper storage and handling of HECW2 antibody, HRP conjugated is critical for maintaining its activity and specificity. The antibody is typically supplied in liquid format with appropriate preservatives . For optimal performance and longevity, the antibody should be stored according to manufacturer recommendations, typically at 2-8°C for short-term storage (1-2 weeks) and at -20°C for long-term storage, preferably in small aliquots to avoid repeated freeze-thaw cycles . When handling the antibody, researchers should wear appropriate personal protective equipment and avoid contamination. The HRP conjugate is sensitive to certain preservatives and metallic surfaces, so plastic tubes and pipette tips are recommended for handling. Additionally, exposure to strong light should be minimized as it may affect the HRP enzyme activity.

How should I optimize ELISA protocols when using HECW2 antibody, HRP conjugated?

Optimizing ELISA protocols with HECW2 antibody, HRP conjugated requires methodical adjustment of several parameters to achieve maximum sensitivity and specificity. Begin with the manufacturer's recommended dilution (typically 1:1000) and modify based on your specific experimental conditions. The following methodological approach is recommended:

• Coating optimization: Test different coating buffer compositions (carbonate/bicarbonate buffer pH 9.6 vs. PBS pH 7.4) and concentrations of capture antibody or antigen.

• Blocking optimization: Evaluate different blocking agents (BSA, non-fat milk, commercial blockers) at various concentrations (1-5%) and incubation times (1-2 hours at room temperature or overnight at 4°C).

• Antibody dilution: Perform a titration series (e.g., 1:500, 1:1000, 1:2000, 1:5000) to determine the optimal dilution that provides maximum signal with minimal background.

• Substrate selection: HRP-conjugated antibodies can be detected using different substrates (TMB, ABTS, OPD), each with different sensitivity profiles. Testing multiple substrates can help identify the optimal choice for your specific application.

• Incubation conditions: Optimize temperature (room temperature vs. 37°C) and duration (30 minutes to 2 hours) for antibody binding to maximize signal while minimizing non-specific interactions.

When validating the ELISA, include positive and negative controls, as well as samples with known HECW2 expression levels to establish a standard curve . This methodical optimization approach will ensure robust and reproducible results.

What controls should be included when studying HECW2 in experimental models?

When studying HECW2 in experimental models, especially in cancer progression or neurological disorder contexts, appropriate controls are essential for valid data interpretation. The following controls should be considered:

• Antibody specificity controls:

  • Peptide competition assay to confirm epitope specificity

  • HECW2 knockout/knockdown samples as negative controls

  • Recombinant HECW2 protein as a positive control

• Experimental controls for HECW2 functional studies:

  • HECW2 knockdown controls: When studying colorectal cancer models, include both HECW2 knockdown and control cells to evaluate changes in proliferation, migration, and chemoresistance

  • HECW2 overexpression controls: Include appropriate vector-only controls alongside HECW2 overexpression models

  • Pathway inhibitor controls: When studying HECW2's role in AKT/mTOR signaling, include AKT inhibitors to confirm pathway specificity

• Tissue-specific controls:

  • For neuronal studies, include both affected and unaffected brain regions

  • For cancer studies, include matched normal tissue alongside tumor samples

  • For developmental studies, include appropriate age-matched controls

How can I verify the specificity of HECW2 antibody, HRP conjugated in my samples?

Verifying antibody specificity is crucial for reliable research outcomes. For HECW2 antibody, HRP conjugated, consider these methodological approaches:

• Western blot validation:

  • Confirm a single band of appropriate molecular weight (~197 kDa for full-length HECW2)

  • Compare with recombinant HECW2 protein (495-641AA) as a positive control

  • Include HECW2 knockdown/knockout samples as negative controls

• Peptide competition assay:

  • Pre-incubate the antibody with excess immunizing peptide

  • Run parallel assays with blocked and unblocked antibody

  • Specific binding should be abolished or significantly reduced in the presence of competing peptide

• Cross-reactivity assessment:

  • Test the antibody on samples from different species (human and mouse)

  • Verify that reactivity matches the manufacturer's specifications

  • For human-specific antibodies, include mouse samples as negative controls and vice versa

• Immunoprecipitation followed by mass spectrometry:

  • Perform IP with the HECW2 antibody

  • Analyze pulled-down proteins by mass spectrometry

  • Confirm HECW2 as the predominant protein identified

These validation steps should be performed before using the antibody for critical experiments, particularly in cases where HECW2 expression or function is central to the research question.

How can HECW2 antibody, HRP conjugated be used to investigate HECW2's role in cancer progression?

Recent research has implicated HECW2 in cancer progression, particularly in colorectal cancer (CRC), where it promotes tumor growth and chemoresistance . To investigate HECW2's role in cancer using HRP-conjugated antibodies, consider these methodological approaches:

• Expression profiling in clinical samples:

  • Use ELISA to quantify HECW2 levels in patient serum or tissue lysates

  • Compare expression between normal, pre-cancerous, and cancerous tissues

  • Correlate expression levels with clinical parameters (stage, grade, treatment response)

• Mechanistic studies of AKT/mTOR pathway activation:

  • Develop sandwich ELISA systems to measure both HECW2 and phosphorylated AKT/mTOR

  • Compare pathway activation in HECW2-high vs. HECW2-low samples

  • Use ELISA to track changes in signaling dynamics after drug treatment

• Chemoresistance assessment:

  • Monitor HECW2 expression changes in response to chemotherapy using ELISA

  • Compare HECW2 levels between chemosensitive and chemoresistant cell populations

  • Use as a biomarker to predict treatment response in patient-derived samples

Research has shown that HECW2 knockdown inhibits CRC progression and chemoresistance, while overexpression has the opposite effect. HECW2 activates the AKT/mTOR signaling pathway by mediating ubiquitin-proteasome degradation of lamin B1 . This mechanism can be further investigated using antibody-based approaches to monitor both HECW2 expression and its downstream effects on target proteins and signaling pathways.

What considerations are important when studying HECW2 in neurological disorders?

HECW2 has been implicated in neurological disorders, with mutations associated with intellectual disability and epilepsy . When studying HECW2 in this context using HRP-conjugated antibodies, several important considerations must be addressed:

• Mutation-specific considerations:

  • De novo mutations in HECW2 have been identified in patients with intellectual disability and epilepsy

  • Many of these mutations cluster in the HECT domain, which is critical for ubiquitin ligase activity

  • Consider whether your antibody's epitope (e.g., AA 495-641) is affected by known mutations

• Tissue-specific expression analysis:

  • Different brain regions may express varying levels of HECW2

  • Use ELISA to quantify region-specific expression patterns in normal and pathological samples

  • Consider developmental timing, as HECW2 expression may change during neuronal maturation

• Functional pathway analysis:

  • HECW2 may interact with other proteins implicated in neurodevelopmental disorders

  • Consider dual-detection approaches to simultaneously measure HECW2 and interacting partners

  • Investigate ubiquitination targets in neuronal contexts using ELISA-based approaches

• Animal model considerations:

  • When using mouse models, ensure the antibody has cross-reactivity with mouse HECW2

  • Consider the specific mutation being modeled and whether it affects antibody binding

  • Include appropriate controls for developmental stage-specific analyses

Studies have shown that HECW2 mutations can disrupt protein function, potentially affecting neuronal development and synaptic function . Using HRP-conjugated antibodies in carefully designed experiments can help elucidate the specific mechanisms by which HECW2 dysfunction contributes to neurological disorders.

How can I investigate HECW2's E3 ubiquitin ligase activity and its targets?

As an E3 ubiquitin ligase, HECW2 mediates the transfer of ubiquitin to specific substrate proteins, including TP73, thereby regulating their stability and function . Investigating this activity requires specialized approaches:

• Detection of ubiquitination targets:

  • Develop sandwich ELISA systems using HECW2 antibody, HRP conjugated in combination with antibodies against known or suspected targets

  • Measure both total protein levels and ubiquitinated fractions

  • Compare ubiquitination patterns in systems with normal vs. altered HECW2 activity

• Domain-specific investigations:

  • HECW2 contains multiple functional domains including HECT, C2, and WW domains

  • The HECT domain (containing the antibody epitope AA 495-641) is particularly important for catalytic activity

  • Use antibodies targeting different domains to investigate domain-specific functions

• Target identification approaches:

  • Combine immunoprecipitation with mass spectrometry to identify novel HECW2 substrates

  • Develop ELISA systems to validate and quantify interactions with newly identified targets

  • Investigate alterations in target protein levels following HECW2 modulation

The following table summarizes key HECW2 domains and their functions that can be investigated using specific antibodies:

Research has demonstrated that HECW2 can interact with AKT in colorectal cancer cells, although this interaction appears to be ubiquitination-independent . This highlights the importance of investigating both canonical and non-canonical functions of HECW2 in different biological contexts.

Why might I be getting weak signal when using HECW2 antibody, HRP conjugated in ELISA?

Weak signal when using HECW2 antibody, HRP conjugated in ELISA can stem from multiple sources. Here's a methodical approach to troubleshooting:

• Antibody activity issues:

  • HRP enzyme activity may be compromised by improper storage or handling

  • Verify activity using a simple dot blot with substrate on a membrane

  • Consider using freshly prepared aliquots to avoid freeze-thaw degradation

• Protocol optimization parameters:

  • Antibody concentration: Try higher concentrations (e.g., 1:500 instead of 1:1000)

  • Incubation conditions: Extend antibody incubation time or adjust temperature

  • Substrate development: Extend development time or try alternative HRP substrates with higher sensitivity

• Target protein considerations:

  • Low HECW2 expression in your samples may result in weak signals

  • Verify HECW2 expression using alternative methods (e.g., Western blot, qPCR)

  • Consider using a more sensitive detection system or sample concentration methods

• Technical factors:

  • Buffer composition may affect antibody binding efficiency

  • Plate coating efficiency may be suboptimal

  • Blocking conditions might be too stringent

A systematic approach to identifying and addressing these factors will help optimize your ELISA protocol for detecting HECW2 using HRP-conjugated antibodies.

How can I reduce background when using HECW2 antibody, HRP conjugated?

High background is a common challenge when using HRP-conjugated antibodies in ELISA and other applications. Here are methodological approaches to reduce background:

• Blocking optimization:

  • Test different blocking agents (BSA, non-fat milk, commercial blockers)

  • Extend blocking time (2 hours at room temperature or overnight at 4°C)

  • Consider adding 0.1-0.3% Tween-20 to blocking and washing buffers

• Washing protocol enhancement:

  • Increase washing stringency (more wash cycles, longer duration)

  • Use freshly prepared washing buffers with appropriate detergent concentration

  • Consider automated washing for more consistent results

• Antibody dilution and diluent optimization:

  • Prepare antibody in blocking buffer containing 0.1% Tween-20

  • Test higher dilutions (e.g., 1:2000 or 1:5000) if signal-to-noise ratio permits

  • Consider adding carrier proteins (BSA, non-fat milk) to antibody diluent

• Substrate considerations:

  • Reduce substrate incubation time if background develops too quickly

  • Consider alternative substrates with different kinetic properties

  • Optimize stopping conditions to prevent overdevelopment

• Sample-specific treatments:

  • Pre-absorb samples with appropriate controls if cross-reactivity is suspected

  • Consider pre-clearing complex samples before analysis

  • Validate samples with known HECW2 expression patterns

These methodological adjustments should be approached systematically, changing one variable at a time and documenting the effects on both signal and background levels.

What are the best practices for data interpretation when using HECW2 antibody, HRP conjugated?

• Standard curve establishment:

  • Create a standard curve using recombinant HECW2 protein (495-641AA)

  • Ensure the curve covers the expected range of HECW2 concentrations in your samples

  • Verify linearity within the working range (R² > 0.95)

• Controls interpretation:

  • Include positive controls (samples with known HECW2 expression)

  • Include negative controls (HECW2 knockout/knockdown samples)

  • Use technical replicates (minimum of triplicates) to assess reproducibility

• Normalization approaches:

  • For comparative studies, normalize HECW2 expression to appropriate housekeeping proteins

  • Consider total protein normalization approaches for more accurate quantification

  • Account for background signal through proper blank subtraction

• Statistical considerations:

  • Apply appropriate statistical tests based on data distribution

  • Consider biological significance alongside statistical significance

  • Acknowledge limitations in interpretation, particularly for novel findings

• Validation with orthogonal methods:

  • Confirm key findings using alternative detection methods (Western blot, immunofluorescence)

  • Validate functional implications through genetic or pharmacological manipulation

  • Consider protein-protein interaction studies to confirm mechanistic hypotheses

By following these best practices, researchers can ensure robust and reproducible data interpretation when using HECW2 antibody, HRP conjugated in their studies.

How is HECW2 antibody, HRP conjugated being used in cancer biomarker research?

HECW2 is emerging as a potential biomarker in cancer research, particularly in colorectal cancer where it has been implicated in progression and chemoresistance . HECW2 antibody, HRP conjugated offers specific advantages for biomarker development:

• Clinical sample screening applications:

  • Development of high-throughput ELISA screening for HECW2 in patient samples

  • Correlation of HECW2 levels with treatment response and patient outcomes

  • Integration with other biomarkers to create predictive panels

• Therapeutic response monitoring:

  • Tracking changes in HECW2 expression during treatment

  • Identifying resistance mechanisms associated with HECW2 upregulation

  • Guiding personalized treatment decisions based on HECW2 status

• Multi-parameter analysis:

  • Combining HECW2 detection with other markers in the AKT/mTOR pathway

  • Creating pathway activation signatures that include HECW2

  • Developing decision algorithms based on multiple biomarkers

Recent research has demonstrated that HECW2 is highly expressed in colorectal cancer tissues and cells, and its expression correlates with disease progression and treatment resistance . The ability to reliably quantify HECW2 using HRP-conjugated antibodies in ELISA formats provides researchers with a valuable tool for investigating its potential as a biomarker in cancer and potentially other diseases.

What emerging techniques are being combined with HECW2 antibody, HRP conjugated for advanced research?

Researchers are combining HECW2 antibody, HRP conjugated with emerging techniques to advance understanding of HECW2 biology:

• High-throughput screening applications:

  • Microarray-based ELISA for screening multiple samples simultaneously

  • Automated liquid handling systems for increased reproducibility

  • Machine learning algorithms for pattern recognition in complex datasets

• Single-cell analysis approaches:

  • Flow cytometry combining HECW2 detection with other cellular markers

  • Single-cell western blotting for heterogeneity analysis

  • Mass cytometry for multi-parameter single-cell profiling

• Spatial biology integration:

  • Combining HECW2 detection with spatial transcriptomics

  • Correlating HECW2 protein levels with local gene expression patterns

  • Mapping HECW2 distribution in complex tissues like brain or tumors

• Systems biology approaches:

  • Network analysis integrating HECW2 with interacting proteins

  • Pathway modeling to predict effects of HECW2 modulation

  • Multi-omics integration connecting HECW2 to broader biological processes

These emerging techniques allow researchers to move beyond simple detection of HECW2 to understanding its functional role in complex biological systems, particularly in neurological disorders and cancer, where HECW2 has been implicated in disease mechanisms .

How might HECW2 research evolve based on recent findings in neurological disorders?

The identification of HECW2 mutations in patients with intellectual disability and epilepsy opens new research directions where HECW2 antibody, HRP conjugated will play an important role :

• Mutation-specific functional analysis:

  • Developing assays to measure the functional impact of specific HECW2 mutations

  • Comparing wildtype and mutant HECW2 activity in neuronal contexts

  • Identifying mutation-specific effects on protein-protein interactions

• Developmental neurobiology applications:

  • Tracking HECW2 expression during critical neurodevelopmental windows

  • Correlating HECW2 levels with neuronal maturation markers

  • Investigating HECW2's role in synapse formation and maintenance

• Therapeutic target validation:

  • Screening compounds that modulate HECW2 activity or expression

  • Developing high-throughput assays for drug discovery

  • Validating targets downstream of HECW2 for therapeutic intervention

• Genotype-phenotype correlation studies:

  • Quantifying HECW2 expression in patient-derived samples

  • Correlating protein levels with severity of neurological symptoms

  • Identifying potential biomarkers for disease progression or treatment response

Previous studies have noted that HECW2 mutations cluster in the HECT domain, which is crucial for ubiquitin ligase activity . This suggests that disruption of HECW2's enzymatic function may be a key mechanism underlying neurological disorders, providing a specific focus for future research using HECW2 antibodies and related tools.