WWP2 Antibody, HRP conjugated

What is WWP2 and why is it important in research?

WWP2 (also known as AIP2) is a WW domain containing E3 ubiquitin protein ligase with significant biological functions. In humans, the canonical protein consists of 870 amino acid residues with a molecular mass of 98.9 kDa and primarily localizes to the nucleus. It plays crucial roles in the regulation of transcription and gene expression through its ubiquitin ligase activity. WWP2 is broadly expressed in fetal tissues including brain, placenta, lung, liver, muscle, kidney, and pancreas. Its involvement in protein degradation pathways makes it a valuable target for studying various cellular processes and disease mechanisms .

What are the key isoforms of WWP2 that can be detected by antibodies?

Up to four different isoforms of WWP2 have been reported in scientific literature. When selecting an antibody, researchers should consider which isoform(s) they need to detect based on their experimental objectives. The commonly used WWP2 antibodies are designed to recognize epitopes that may be present in all isoforms or specific to particular variants. For comprehensive studies, researchers often use antibodies targeting conserved domains, while isoform-specific investigations require more specialized reagents targeting unique regions . The epitope location (N-terminal, middle region, or C-terminal) determines which isoforms will be detected in experimental applications.

What are the common applications for WWP2 HRP-conjugated antibodies?

WWP2 HRP-conjugated antibodies are primarily used in Western Blot (WB), Enzyme-Linked Immunosorbent Assay (ELISA), and immunohistochemistry on paraffin-embedded tissues (IHC-p). The direct HRP conjugation eliminates the need for secondary antibody incubation, reducing background noise and protocol time. This is particularly advantageous in multiplex protein detection experiments where antibodies from the same host species need to be used simultaneously . Published literature shows these antibodies can effectively detect WWP2 expression levels, post-translational modifications, and protein-protein interactions in various experimental contexts.

How do I optimize my WWP2 antibody dilution for Western blot experiments?

Optimization of WWP2 HRP-conjugated antibody dilution is critical for obtaining clear results with minimal background. Start with the manufacturer's recommended dilution (typically 1:1000 to 1:5000 for Western blot applications) and perform a dilution series experiment using positive control samples. When working with HRP-conjugated WWP2 antibodies, remember that excessive antibody concentration can lead to high background, while insufficient amounts may result in weak signals. Document signal-to-noise ratio at each dilution under standardized exposure conditions. For Santa Cruz AIP2 (A-3) HRP Antibody with concentration of 200 μg/ml, researchers typically start with dilutions between 1:500 and 1:2000 for optimal results . Always include appropriate positive and negative controls to validate specificity.

What are the advantages and limitations of using WWP2 HRP-conjugated antibodies compared to unconjugated versions?

The decision should be based on specific experimental needs, with HRP-conjugated versions being particularly valuable for multiplex detection systems and rapid protocols .

How do I troubleshoot weak or absent signals when using WWP2 HRP-conjugated antibodies in Western blotting?

When encountering weak or absent signals with WWP2 HRP-conjugated antibodies, a systematic troubleshooting approach is necessary. First, verify sample integrity by confirming protein concentration and checking for degradation with Ponceau S staining. Next, evaluate transfer efficiency using reversible total protein stains. For WWP2-specific issues, consider that different lysis buffers may be required to efficiently extract nuclear proteins where WWP2 predominantly localizes. Increasing sample amount (50-100 μg total protein) may be necessary for detecting endogenous WWP2, as noted in user feedback regarding Santa Cruz WWP2 antibodies . Also confirm appropriate blocking conditions (5% BSA often performs better than milk for phosphorylated targets) and ensure fresh detection reagents. Finally, remember that the 98.9 kDa WWP2 protein may require longer transfer times (60-90 minutes) than smaller proteins for complete membrane transfer .

What is the recommended protocol for detecting WWP2 in different subcellular fractions?

To effectively detect WWP2 in different subcellular fractions, a specialized fractionation and detection protocol is recommended:

• Perform subcellular fractionation using established protocols to separate nuclear, cytoplasmic, and membrane fractions

• Include appropriate fraction-specific markers for validation (e.g., Lamin B1 for nuclear fraction)

• Load 30-50 μg of protein from each fraction for SDS-PAGE

• Transfer to PVDF membrane (preferred over nitrocellulose for WWP2 detection)

• Block with 5% BSA in TBST for 1 hour at room temperature

• Incubate with WWP2 HRP-conjugated antibody at 1:1000 dilution overnight at 4°C

• Wash extensively with TBST (5 × 5 minutes)

• Develop using ECL substrate with exposure times optimized for each fraction

This protocol accounts for the nuclear localization of canonical WWP2 while also detecting other isoforms that may distribute differently within cells . The approach enables researchers to investigate compartment-specific functions and regulations of WWP2 in different cellular contexts.

What are the best experimental conditions for immunoprecipitation using WWP2 antibodies?

For successful immunoprecipitation (IP) of WWP2, several critical parameters must be optimized:

First, cell lysis conditions significantly impact WWP2 recovery. A recommended lysis buffer composition includes 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, with freshly added protease inhibitors and, importantly, deubiquitinase inhibitors such as N-ethylmaleimide (10 mM) to preserve ubiquitination states. Since WWP2 functions in ubiquitin-mediated pathways, preserving these modifications is crucial for interaction studies.

For the IP procedure itself, pre-clearing lysates with protein A/G beads (1 hour at 4°C) reduces non-specific binding. According to published protocols using Bethyl Laboratories' WWP2 antibodies, 2-5 μg of antibody per 500 μg of total protein yields optimal results . When using agarose-conjugated WWP2 antibodies like the Santa Cruz AIP2 (A-3) AC antibody, 20-40 μl of bead slurry per sample provides efficient capture .

Notably, IP efficiency may vary between human and mouse samples, with some antibodies showing stronger performance with human WWP2. Always validate IP conditions with both positive controls and IgG negative controls to ensure specificity.

How do different fixation methods affect WWP2 detection in immunohistochemistry?

The choice of fixation method significantly impacts WWP2 epitope preservation and detection sensitivity in immunohistochemistry. A comparative analysis of different fixation protocols reveals:

Formalin fixation (10% neutral buffered formalin, 24 hours) provides good morphological preservation but requires robust antigen retrieval for WWP2 detection. Heat-induced epitope retrieval in citrate buffer (pH 6.0) for 20 minutes typically yields optimal results with most WWP2 antibodies.

Paraformaldehyde fixation (4% PFA, 12-24 hours) offers a good balance between morphology and antigen preservation, making it suitable for most WWP2 detection protocols, particularly when studying nuclear localization patterns.

Methanol fixation (-20°C, 10 minutes) can enhance detection of certain WWP2 epitopes while potentially compromising others, especially in the WW domains. This fixative works well for detecting cytoplasmic WWP2 interactions but may not preserve nuclear WWP2 optimally.

Acetone fixation (ice-cold, 10 minutes) provides superior detection of some conformational epitopes but with reduced morphological quality, making it less suitable for detailed localization studies.

For HRP-conjugated WWP2 antibodies, additional considerations include thorough quenching of endogenous peroxidase activity (3% H₂O₂, 10 minutes) prior to antibody application to minimize background . This comprehensive approach to fixation optimization ensures reliable WWP2 detection across diverse tissue types.

What controls should be included when studying WWP2 in experimental systems?

A robust experimental design for studying WWP2 requires multiple controls to ensure data validity and interpretability:

Positive controls: Include cell lines or tissues known to express WWP2 at detectable levels. Based on published research, HEK293, MCF7, and A549 cell lines consistently show moderate to high WWP2 expression levels and serve as reliable positive controls .

Negative controls:

• Primary antibody omission: Process samples with all reagents except the WWP2 antibody

• Isotype control: Use matched isotype antibody at the same concentration

• Genetic controls: When available, include WWP2 knockout/knockdown samples

• Peptide competition: Pre-absorb antibody with immunizing peptide before application

Specificity controls:

• Test for cross-reactivity with related E3 ligases, particularly other NEDD4 family members

• When studying specific isoforms, include controls that express only the isoform of interest

Application-specific controls:

• For Western blot: Include molecular weight markers and loading controls (e.g., β-actin)

• For IHC/ICC: Include tissues/cells with known expression patterns

• For IP experiments: Include IgG control pulls to identify non-specific binding

This comprehensive control strategy minimizes the risk of experimental artifacts and ensures reproducible, interpretable results when studying WWP2 biology .

How should I analyze WWP2 expression data across different experimental models?

When analyzing WWP2 expression across different experimental models, researchers should employ a structured approach to ensure comparable and reliable results:

First, normalize WWP2 expression to appropriate reference standards. For Western blot analyses, normalization to housekeeping proteins like β-actin or GAPDH is standard, though nuclear loading controls like Lamin B1 may be more appropriate given WWP2's nuclear localization. For qRT-PCR data, use multiple reference genes (GAPDH, ACTB, and RPL13A) for more robust normalization.

Second, quantify relative WWP2 levels using digital image analysis software with standardized background subtraction methods. For Western blots, integrated density values provide more reliable quantification than band intensity alone. Report WWP2 expression as fold-change relative to control conditions rather than absolute values.

Third, account for isoform-specific expression patterns. The canonical 98.9 kDa WWP2 form may show different regulation than smaller isoforms in the 50-70 kDa range. Document all detected bands and validate their identity through appropriate controls.

Finally, perform statistical analysis appropriate to your experimental design. For comparing WWP2 expression across multiple experimental conditions, ANOVA with post-hoc tests is generally more appropriate than multiple t-tests. Include biological replicates (n≥3) to account for natural variation in WWP2 expression .

How can I study the interaction between WWP2 and its target proteins?

Investigating interactions between WWP2 and its target proteins requires specialized approaches due to the often transient nature of E3 ligase-substrate interactions. A comprehensive strategy includes:

Co-immunoprecipitation with modifications: Standard co-IP protocols often fail to capture WWP2-substrate interactions due to their transient nature. Adding proteasome inhibitors (MG132, 10 μM) and deubiquitinase inhibitors (PR-619, 10 μM) to lysis buffers significantly enhances detection of these interactions. For HRP-conjugated WWP2 antibodies, elution conditions must be optimized to preserve both antibody activity and protein-protein interactions.

Proximity ligation assay (PLA): This technique allows visualization of protein interactions in situ with high sensitivity. For studying WWP2 interactions, use the HRP-conjugated WWP2 antibody in combination with antibodies against suspected interaction partners, followed by appropriate PLA probes. This approach has successfully identified interactions between WWP2 and transcription factors in published studies.

Bimolecular Fluorescence Complementation (BiFC): By fusing WWP2 and potential targets to complementary fragments of fluorescent proteins, researchers can visualize interactions in living cells. This approach is particularly valuable for studying the dynamics of WWP2-substrate interactions.

FRET/FLIM analysis: For more detailed spatial and temporal resolution of WWP2 interactions, Förster Resonance Energy Transfer combined with Fluorescence Lifetime Imaging provides quantitative measurements of protein proximity in living cells.

These methods can be combined with domain mapping approaches (using truncated WWP2 constructs) to identify specific interaction domains within the WWP2 protein structure, providing mechanistic insights into substrate recognition .

What are the key considerations when studying WWP2-mediated ubiquitination?

Studying WWP2-mediated ubiquitination presents several technical challenges that require specific methodological considerations:

Ubiquitination chain specificity: WWP2 can catalyze multiple ubiquitin chain types (K48, K63, K27) depending on the substrate and cellular context. When designing ubiquitination assays, include chain-specific ubiquitin antibodies to determine the precise modification pattern. K48-linked chains typically signal for proteasomal degradation, while K63-linked chains often regulate protein function or localization.

In vitro ubiquitination assays: To establish direct WWP2-mediated ubiquitination, reconstituted systems require purified components including E1 (UBA1), an appropriate E2 (typically UbcH5 family members), recombinant WWP2, ubiquitin, ATP, and the putative substrate. Reaction conditions (pH 7.5, 30°C, 1-2 hours) must be carefully optimized for each substrate.

Cellular ubiquitination assays: When using HRP-conjugated WWP2 antibodies in cellular contexts, include appropriate controls:

• WWP2 catalytic mutants (C838A in the HECT domain)

• Dominant-negative WWP2 constructs

• siRNA/shRNA-mediated WWP2 knockdown

Detection strategies: Denaturing conditions (8M urea or 1% SDS in lysis buffers) are essential to disrupt non-covalent interactions and ensure only covalently attached ubiquitin is detected. Two-step immunoprecipitation protocols (first capturing the substrate, then detecting ubiquitin) provide the most conclusive evidence of substrate-specific ubiquitination.

These methodological refinements help researchers distinguish direct WWP2-mediated ubiquitination from indirect effects and identify the functional consequences of these modifications in various biological contexts .

How do I interpret conflicting WWP2 antibody results across different experimental platforms?

When faced with conflicting WWP2 antibody results across different experimental platforms, a systematic analytical approach is essential:

First, evaluate epitope accessibility in different applications. The WWP2 protein contains multiple domains (C2 domain, four WW domains, and a HECT domain) that may be differentially exposed depending on the application. For example, an antibody targeting the HECT domain might perform well in Western blots where proteins are denatured but poorly in immunoprecipitation where native conformations are maintained.

Second, consider isoform specificity. The four reported WWP2 isoforms have different domain compositions and may be differentially detected depending on the antibody's epitope location. Antibodies targeting the N-terminal region may not detect C-terminal isoforms and vice versa. Cross-reference your results with data from antibodies targeting different regions of the protein.

Third, analyze post-translational modifications. WWP2 undergoes auto-ubiquitination and other modifications that may mask epitopes in certain contexts. Different experimental conditions may preserve or disrupt these modifications, affecting antibody binding.

Fourth, examine species cross-reactivity carefully. Despite high conservation, subtle species-specific differences in WWP2 sequence may impact antibody performance. The reported cross-reactivity with human, mouse, and rat samples should be experimentally verified in your specific model systems.

Finally, implement a multi-antibody validation strategy. When critical results depend on WWP2 detection, use at least two independent antibodies targeting different epitopes. Concordant results significantly increase confidence in your findings .

What are the emerging applications for WWP2 antibodies in cancer research?

WWP2 antibodies are increasingly utilized in cancer research applications, revealing important roles for this E3 ligase in tumor biology:

Prognostic biomarker development: Recent studies have correlated WWP2 expression levels with patient outcomes in several cancer types. Immunohistochemistry using HRP-conjugated WWP2 antibodies on tissue microarrays has identified overexpression patterns associated with aggressive disease phenotypes, particularly in prostate and breast cancers. Standardized scoring systems for WWP2 immunoreactivity are being developed to facilitate clinical implementation.

Therapeutic target validation: As a regulator of multiple oncogenic pathways, WWP2 represents a potential therapeutic target. Antibody-based detection methods are essential for validating WWP2-targeted drug efficacy in preclinical models. Multiplexed immunofluorescence approaches combining WWP2 detection with markers of cell proliferation, apoptosis, and stemness provide comprehensive assessment of therapeutic responses.

Pathway analysis: WWP2 has been implicated in the regulation of TGF-β signaling, PTEN degradation, and OCT4 stability, all critical in cancer development. Co-immunoprecipitation studies using WWP2 antibodies have identified novel interaction partners in cancer-specific contexts, expanding our understanding of WWP2's oncogenic functions.

Resistance mechanism identification: Emerging evidence suggests that altered WWP2 expression or activity may contribute to therapy resistance. Antibody-based screens comparing treatment-sensitive and resistant cells have identified WWP2-dependent pathways that could be targeted to overcome resistance.

These applications highlight the growing importance of high-quality, well-validated WWP2 antibodies in translational cancer research and potential therapeutic development .

What are the key quality control parameters for evaluating WWP2 HRP-conjugated antibodies?

Rigorous quality control is essential for reliable WWP2 HRP-conjugated antibody performance. Key parameters to evaluate include:

Antibody specificity: Validate using Western blot analysis against recombinant WWP2 protein and endogenous WWP2 from appropriate cell lysates. Look for a predominant band at 98.9 kDa (canonical isoform) with potentially additional bands representing known isoforms. Absence of non-specific bands at unexpected molecular weights indicates good specificity.

HRP conjugation efficiency: Measured through enzyme activity assays using standard substrates like TMB or luminol. Optimal conjugation preserves both antibody binding and enzymatic activity. The molar ratio of HRP to antibody should be consistent between batches (typically 2-4 HRP molecules per antibody molecule for optimal performance).

Lot-to-lot consistency: Compare performance across multiple lots using standardized positive controls. Signal intensity should not vary by more than 15-20% between lots when used at identical concentrations under identical conditions.

Stability assessment: Evaluate antibody performance after storage under recommended conditions at defined time points (0, 3, 6, and 12 months). Minimal loss of signal (<30% over 12 months) indicates good stability.

Application-specific validation: For WWP2 HRP-conjugated antibodies, specific validation in their intended applications (WB, ELISA, IHC-p) is essential, as performance can vary significantly between applications even for the same antibody.

Background evaluation: Measure signal-to-noise ratio under standardized conditions. High-quality antibodies typically show signal-to-background ratios >10:1 in optimized protocols .

How do I determine the optimal antibody concentration for my specific application?

Determining the optimal concentration for WWP2 HRP-conjugated antibodies requires a systematic titration approach tailored to each application:

For Western blotting, begin with a broad range titration using 2-fold serial dilutions from 1:500 to 1:8000 of the antibody (starting from standard concentration of 200 μg/ml as seen with Santa Cruz AIP2 HRP antibody) . Use positive control samples with known WWP2 expression. The optimal dilution provides strong specific signal with minimal background under standardized exposure conditions.

For immunohistochemistry, a more narrow initial range is recommended (1:50 to 1:500), as tissue applications typically require higher antibody concentrations. The optimal dilution should produce specific staining of appropriate subcellular localization (primarily nuclear for WWP2) without background or edge artifacts.

For ELISA applications, construct a complete standard curve using recombinant WWP2 protein at known concentrations. Test antibody dilutions ranging from 1:1000 to 1:10,000, selecting the concentration that provides the widest dynamic range while maintaining sensitivity to detect your expected concentration range.

Important considerations for optimization include:

• Sample type (cell lysates vs. tissue sections)

• Expression level (endogenous vs. overexpressed)

• Detection method (chemiluminescence sensitivity varies by substrate)

• Incubation conditions (overnight at 4°C often improves signal-to-noise ratio compared to 1-2 hours at room temperature)

Document optimal conditions in a detailed protocol to ensure reproducibility across experiments .

How do WWP2 antibodies from different suppliers compare in research applications?

A comparative analysis of WWP2 antibodies from various suppliers reveals significant performance differences across research applications:

Western Blot Performance: Based on user feedback and published citations, Bethyl Laboratories' WWP2 antibodies consistently show strong performance in Western blot applications, with clear detection of the 98.9 kDa canonical form and minimal background. These antibodies have accumulated multiple citations (7) validating their specificity . In contrast, some researchers have reported inconsistent results with Santa Cruz WWP2 antibodies for detecting endogenous protein, though they perform well with overexpressed systems .

Immunoprecipitation Efficiency: For IP applications, Bethyl and Abcam antibodies demonstrate superior performance with published validation. Specifically, Abcam's anti-WWP2 antibody has gathered 12 citations supporting its use in IP experiments . The agarose-conjugated Santa Cruz antibody (AIP2 A-3 AC) offers the advantage of direct immunoprecipitation without requiring separate capture beads but may have more limited epitope recognition.

Immunohistochemistry Applications: For IHC, antibodies targeting the middle region of WWP2, such as those from Aviva Systems Biology (ARP43088), show better performance in paraffin-embedded tissues after appropriate antigen retrieval. These antibodies detect WWP2 across multiple species including human, mouse, and rat models .

Species Cross-Reactivity: While most suppliers claim multi-species reactivity, actual performance varies significantly. Cell Signaling's WWP2 (E8W8A) Rabbit mAb shows high specificity for human samples but limited cross-reactivity with rodent models, making it ideal for human cell line research but less suitable for mouse studies .

This comparative analysis highlights the importance of selecting WWP2 antibodies based on the specific application, species, and experimental system rather than relying solely on supplier claims or antibody format .

What criteria should inform the selection of a WWP2 HRP-conjugated antibody for specific research applications?

Selecting the optimal WWP2 HRP-conjugated antibody requires evaluation of multiple criteria tailored to specific research applications:

Epitope location: Consider where the antibody binds on the WWP2 protein. N-terminal antibodies detect most isoforms but may be affected by protein interactions, while C-terminal antibodies specifically recognize the full-length canonical form. For studying specific WWP2 functions, select antibodies targeting functional domains (WW or HECT domains) relevant to your research question.

Clonality considerations: Monoclonal antibodies like Cell Signaling's E8W8A offer excellent batch-to-batch consistency and specificity for a single epitope, making them ideal for quantitative applications. Polyclonal antibodies from providers like Bethyl Laboratories provide broader epitope recognition, potentially increasing sensitivity for detection of modified or partially degraded WWP2.

Validation evidence: Prioritize antibodies with published validation data in applications matching your experimental design. The Abcam anti-WWP2 antibody with 12 citations and multiple validation figures represents a well-characterized option . Request validation data specifically showing detection of endogenous (not just overexpressed) WWP2.

HRP conjugation quality: Evaluate the conjugation chemistry used and the enzyme:antibody ratio, as these factors significantly impact sensitivity and stability. Direct HRP conjugation is particularly valuable for multiplex experiments where secondary antibody cross-reactivity is problematic.

Application-specific optimization: For Western blotting, sensitivity to denatured protein is paramount. For IP applications, native epitope recognition is critical. For high-throughput applications like ELISA, consistency and low background are essential.

This systematic evaluation approach ensures selection of the most appropriate WWP2 HRP-conjugated antibody for specific research objectives, improving experimental outcomes and data reliability .

What emerging technologies are enhancing WWP2 antibody applications in research?

Novel technological approaches are expanding the utility and applications of WWP2 antibodies in cutting-edge research. Proximity-based labeling techniques like BioID and APEX2 are being combined with WWP2 antibodies to map the dynamic WWP2 interactome in various cellular contexts. These approaches overcome limitations of traditional co-immunoprecipitation by capturing both stable and transient interactions in living cells.

Advanced microscopy techniques including super-resolution imaging (STORM, PALM) coupled with WWP2 antibody detection are revealing previously unobservable subcellular localization patterns and protein interactions at nanometer-scale resolution. These approaches have identified distinct WWP2 localization patterns within subnuclear structures that correlate with specific cellular functions.

Single-cell proteomics methods incorporating WWP2 antibodies are enabling researchers to examine cell-to-cell variability in WWP2 expression and activity within heterogeneous populations. This is particularly valuable in cancer research, where cellular heterogeneity presents significant challenges to understanding disease mechanisms.

CRISPR-based genomic tagging combined with WWP2 antibody validation is facilitating studies of endogenous WWP2 dynamics without overexpression artifacts. This approach ensures physiologically relevant observations of WWP2 function in various model systems.

Together, these emerging technologies are significantly advancing our understanding of WWP2 biology and opening new avenues for therapeutic targeting in diseases where WWP2 plays critical regulatory roles .

How should researchers address the challenges of reproducibility when working with WWP2 antibodies?

Addressing reproducibility challenges with WWP2 antibodies requires implementation of rigorous validation protocols and standardized reporting practices:

First, implement comprehensive antibody validation that includes: (1) Genetic approaches using WWP2 knockout/knockdown models; (2) Orthogonal methods comparing antibody-based detection with MS-based proteomics; (3) Independent antibody validation using multiple antibodies targeting different epitopes; and (4) Expression pattern analysis confirming expected tissue/cellular distribution.

Second, standardize experimental protocols across research groups. Detailed reporting of antibody information (catalog number, lot number, dilution, incubation conditions) is essential. For WWP2 HRP-conjugated antibodies, additionally report the detection system sensitivity, exposure parameters, and image acquisition settings used for analysis.

Third, establish positive control reference standards. Cell lines with well-characterized WWP2 expression (such as HEK293 or MCF7) should be included as inter-laboratory reference standards. Consider developing shared plasmid repositories for WWP2 expression constructs to ensure consistent positive controls.

Fourth, implement quantitative reporting standards. Replace subjective descriptions ("strong expression") with quantitative metrics (relative expression normalized to standards). For IHC applications, use established scoring systems with multiple independent evaluators.

Finally, maintain detailed records of antibody performance across experiments to track potential lot-to-lot variations. When publishing results, include key validation data in supplementary materials to support reproducibility.