WISP2 Antibody, HRP conjugated

What are the optimal dilutions and applications for WISP2 antibodies?

For research applications using WISP2 antibodies, optimal dilutions vary by technique. Western blot analysis typically requires a 1:1000 dilution of primary WISP2 antibody followed by appropriate HRP-conjugated secondary antibody for chemiluminescent detection . For immunohistochemistry on paraffin-embedded tissues (IHC-p), dilutions of 1:50-100 are recommended for optimal staining with minimal background . When performing immunohistochemistry, pressure cooking sections in 10 mmol/L EDTA (pH 8.0) for 3 minutes followed by room temperature incubation with WISP2 antibody for 3 hours provides robust results . For co-immunoprecipitation studies examining WISP2 acetylation status, researchers should adjust protocols to preserve post-translational modifications during cell lysis and protein extraction procedures . Researchers should always validate antibody performance in their specific experimental system, as optimal conditions may vary based on tissue type, fixation method, and detection system.

How should researchers validate the specificity of WISP2 antibodies?

Validating WISP2 antibody specificity is crucial for accurate experimental outcomes. Researchers should employ multiple complementary approaches, including positive and negative controls in each experiment. Normal human mammary epithelium serves as an effective positive control for WISP2 detection, while normal gastric mucosa stained with PBS instead of primary antibody provides a suitable negative control . WISP2 knockdown models created using ribozyme transgenes or other gene silencing approaches offer excellent specificity controls, as demonstrated in studies with gastric cancer and breast cancer cell lines . Western blot analysis should reveal bands of appropriate molecular weight (approximately 27 kDa), and researchers should normalize against housekeeping proteins such as GAPDH (1:5000 dilution) for quantitative comparisons . Cross-validation using different antibody clones or detection methods strengthens confidence in results, particularly when investigating subtle changes in WISP2 expression or modifications.

What sample preparation techniques optimize WISP2 detection?

Proper sample preparation significantly impacts successful WISP2 detection. For tissue sections, formalin-fixed paraffin-embedded (FFPE) samples should be sectioned at 4 μm thickness and mounted on poly-L-lysine-coated glass slides . Complete deparaffinization in xylene followed by rehydration through graded alcohols is essential before immunostaining. Blocking endogenous peroxidase activity with 3% hydrogen peroxide for 15 minutes at room temperature prevents non-specific background when using HRP-conjugated detection systems . For cell lysates in Western blotting applications, sonication in appropriate lysis buffer at 4°C followed by centrifugation preserves protein integrity, with total protein concentrations determined via BCA reagent assay . For studies examining WISP2 acetylation status, avoid deacetylase activity during sample preparation by including deacetylase inhibitors in lysis buffers, as acetylation significantly affects WISP2 stability and function . Quantitative analysis should employ standardized protocols, such as densitometric scanning of Western blot films using software like Image J for reproducible results .

How can researchers investigate WISP2 post-translational modifications?

WISP2 undergoes critical post-translational modifications that significantly impact its stability and function. To investigate WISP2 acetylation status, researchers should employ co-immunoprecipitation (co-IP) with anti-acetyl lysine antibodies followed by Western blotting with WISP2-specific antibodies . This approach revealed that WISP2 acetylation at lysine K6 prevents its degradation in AML cells. Site-directed mutagenesis studies, where lysine residues (such as K6 and K20) are mutated to arginine, help confirm specific acetylation sites and their functional importance . For studying how deacetylation affects WISP2 stability, treatments with deacetylase inhibitors (such as valproic acid, trichostatin A, or the HDAC3-specific inhibitor RGFP966) followed by Western blot analysis can demonstrate changes in WISP2 protein levels . When examining ubiquitination-mediated degradation, cycloheximide chase experiments blocking new protein synthesis reveal how post-translational modifications affect WISP2 protein half-life, while co-IP studies with ubiquitin antibodies identify specific E3 ligases (such as NEDD4) involved in the process . These advanced techniques require careful optimization of lysis conditions to preserve transient modifications and protein-protein interactions.

What approaches enable effective study of WISP2's tumor suppressor mechanisms?

Investigating WISP2's tumor suppressor function demands sophisticated experimental approaches. Researchers should establish stable WISP2 knockdown cell lines using ribozyme transgenes or other gene silencing techniques, followed by comprehensive functional analyses including proliferation, adhesion, migration, and invasion assays . Using both in vitro cellular models and in vivo xenograft approaches provides complementary insights. For example, WISP2 overexpression in leukemia cells (HL-60 and Kasumi-1) suppresses cell proliferation, induces apoptosis, and demonstrates antileukemic effects in animal models . To delineate molecular mechanisms, researchers should examine epithelial-mesenchymal transition (EMT) markers, as WISP2 expression inversely correlates with EMT transcription factors like Twist and Slug . Matrix metalloproteinase (MMP) activity assays reveal how WISP2 regulates invasion capacity, showing that WISP2 suppresses gastric cancer cell metastasis by inhibiting MMPs via JNK and ERK signaling pathways . Cell motility analysis with appropriate inhibitors (such as PLC-γ and JNK small inhibitors) further elucidates signaling pathways controlled by WISP2 . Integration of these approaches with patient survival data strengthens translational relevance of laboratory findings.

How can multiplex detection systems be optimized for WISP2 and related pathways?

Multiplex detection systems allow simultaneous analysis of WISP2 and its interacting partners or downstream effectors. For analyzing WISP2 in relation to cell cycle regulators like Skp2 and p27Kip1, researchers should optimize antibody combinations that function under identical experimental conditions . When designing multiplex immunofluorescence protocols, careful selection of primary antibodies from different host species (e.g., rabbit anti-WISP2 with mouse anti-Skp2) prevents cross-reactivity issues. For chemiluminescent Western blot analysis of multiple proteins, sequential probing with careful stripping protocols between detections maintains signal specificity, or alternatively, dual-color infrared imaging systems can distinguish multiple targets on the same membrane . In tissue samples, double immunohistochemical staining with different chromogens allows visualization of WISP2 alongside other markers. For complex pathway analysis, researchers should consider protein arrays or mass spectrometry-based proteomics approaches following WISP2 immunoprecipitation to identify novel interaction partners. These multiplex approaches require extensive validation to ensure antibody performance is not compromised by the presence of multiple detection reagents.

What strategies overcome challenges in detecting low-abundance WISP2 in clinical samples?

Detecting low-abundance WISP2 in clinical samples presents significant technical challenges. To enhance sensitivity, researchers should implement signal amplification techniques such as tyramide signal amplification (TSA) with HRP-conjugated antibodies, which can increase detection sensitivity by 10-100 fold compared to conventional methods . For bone marrow samples from AML patients, where WISP2 expression is often reduced, enrichment of mononuclear cells prior to analysis improves detection capabilities . In tissue microarrays, optimization of antigen retrieval methods is crucial, with pressure cooking in 10 mmol/L EDTA (pH 8.0) recommended for WISP2 epitope exposure . Quantitative scoring systems defining "negative" (0-20% stained cells) versus "positive" expression (>20% stained cells) provide consistent evaluation metrics for immunohistochemistry . For Western blot applications in samples with low WISP2 expression, concentrating protein lysates and extending exposure times with highly sensitive ECL Plus detection systems enhances visualization of faint bands . In all cases, parallel analysis of high-expressing control samples validates detection protocols and establishes assay sensitivity thresholds.

What is the optimal Western blot protocol for WISP2 detection using HRP-conjugated antibodies?

The optimal Western blot protocol for WISP2 detection begins with effective protein extraction through sonication of cells in lysis buffer at 4°C, followed by centrifugation and quantification using BCA reagent assay . Researchers should separate proteins on 10% SDS-PAGE gels before transferring to nitrocellulose membranes . For immunodetection, block membranes appropriately before incubating with anti-WISP2 primary antibody at 1:200 dilution, optimally using a SNAP-id 2.0 machine or similar platform for consistent results . Follow with HRP-conjugated anti-rabbit secondary antibody at manufacturer-recommended dilutions (typically 1:2000-1:5000). For visualization, use ECL Plus Western Blotting Detection System with exposure times tailored to expression levels . For accurate quantitation, normalize WISP2 signals against GAPDH (1:5000) or other appropriate housekeeping proteins . When comparing acetylation status of WISP2 across experimental conditions, perform parallel blots with acetyl-lysine antibodies following immunoprecipitation with WISP2 antibodies . This approach successfully detected increased WISP2 acetylation following treatment with HDAC inhibitors valproic acid and trichostatin A . For challenging samples, consider concentration steps or enhanced chemiluminescence substrates to improve sensitivity.

How should immunohistochemistry protocols be adapted for robust WISP2 visualization?

For optimal WISP2 immunohistochemistry, prepare formalin-fixed, paraffin-embedded tissues sectioned at 4 μm thickness and mounted on poly-L-lysine-coated glass slides . After deparaffinization in xylene and rehydration through alcohol gradients, block endogenous peroxidase activity with 3% hydrogen peroxide for 15 minutes at room temperature . Critical for WISP2 detection is proper antigen retrieval—pressure cooking sections in 10 mmol/L EDTA (pH 8.0) for 3 minutes provides optimal epitope exposure . Incubate sections with anti-WISP2 antibody at 1:100 dilution for 3 hours at room temperature, followed by HRP-conjugated secondary antibody . Develop slides using diaminobenzidine tetrahydrochloride solution for visualization, then counterstain with hematoxylin, dehydrate in ethanol, and clear with xylene before mounting . Always include normal human mammary epithelium as positive control and normal gastric mucosa with PBS substituted for primary antibody as negative control . For quantitative assessment, define expression as "negative" (0-20% stained cells) or "positive" (>20% stained cells), with independent scoring by at least two observers using light microscopy to ensure reproducibility . This protocol successfully detected differential WISP2 expression in gastric cancer specimens compared to adjacent normal tissues.

What factors contribute to inconsistent results with WISP2 antibodies and how can they be addressed?

Several factors may contribute to inconsistent WISP2 antibody results. Protein stability issues arise because WISP2 undergoes rapid degradation through the ubiquitin-proteasome pathway, particularly when deacetylated . To address this, include proteasome inhibitors (e.g., MG132) and deacetylase inhibitors in lysis buffers when preparing samples. Epitope masking due to post-translational modifications may impair antibody recognition—using multiple antibodies targeting different WISP2 regions helps overcome this limitation . Tissue fixation variability significantly impacts immunohistochemistry results; standardize fixation protocols and validate antibody performance in differently fixed samples. Cross-reactivity with other WISP family members occurs due to structural similarities; confirm antibody specificity using WISP2 knockdown controls or recombinant protein standards . Quantification challenges arise from subjective interpretation; implement standardized scoring systems and use digital image analysis where possible . Sample degradation during storage affects detection; prepare fresh lysates or sections when feasible and store samples appropriately (-80°C for protein lysates). By systematically addressing these factors, researchers can achieve consistent, reproducible WISP2 detection across experimental platforms.

How can researchers optimize co-immunoprecipitation for studying WISP2 interactions?

Optimizing co-immunoprecipitation (co-IP) for WISP2 interaction studies requires careful consideration of experimental conditions. Begin with gentle cell lysis using buffers containing 1% NP-40 or similar mild detergents to preserve protein-protein interactions, while including protease inhibitors, phosphatase inhibitors, and deacetylase inhibitors (such as trichostatin A) to maintain post-translational modifications . Pre-clear lysates with appropriate control IgG and protein A/G beads to reduce non-specific binding. For WISP2 acetylation studies, immunoprecipitate with anti-acetyl lysine antibody followed by Western blotting with WISP2 antibody—this approach successfully confirmed enhanced WISP2 acetylation following HDAC inhibitor treatment . Alternatively, immunoprecipitate with WISP2-specific antibody followed by Western blotting for interacting partners or modifications. When studying WISP2 interactions with ubiquitination machinery like NEDD4, include deubiquitinase inhibitors in lysis buffers and consider using lysine-to-arginine mutants (K6R, K20R) to map specific modification sites . Controls should include IgG-matched antibodies, input samples (typically 5-10% of lysate used for IP), and when possible, samples with WISP2 knockdown or overexpression to confirm specificity. Crosslinking reagents can stabilize transient interactions before cell lysis for detection of weak or dynamic interaction partners.

What are the best approaches for analyzing WISP2 in relation to cellular signaling pathways?

Analyzing WISP2 in cellular signaling contexts requires integrated experimental approaches. For examining WISP2 regulation of epithelial-mesenchymal transition (EMT), quantify expression of recognized EMT markers using Q-PCR in paired tumor and normal tissues alongside WISP2 levels . This approach revealed inverse correlations between WISP2 and EMT transcription factors Twist and Slug in gastric cancer samples . To investigate WISP2's impact on cell motility pathways, create WISP2 knockdown cell lines using ribozyme transgenes, then assess changes in migration and invasion while treating with pathway-specific inhibitors (PLC-γ and JNK inhibitors effectively attenuated enhanced motility in WISP2-knockdown gastric cancer cells) . For elucidating relationships between WISP2 and cell cycle regulators, perform correlation analysis at both mRNA and protein levels—this strategy revealed significant associations between WISP2, Skp2, and p27Kip1 in breast cancer models . When analyzing HDAC-mediated regulation of WISP2, treat cells with specific HDAC inhibitors (RGFP966 for HDAC3) before assessing WISP2 protein stability through cycloheximide chase experiments . These comprehensive approaches established that acetylation of WISP2 at lysine K6 increases protein stability by decreasing ubiquitination and proteasomal degradation, with significant implications for cancer progression .

How does WISP2 function differ between cancer types and what detection considerations apply?

WISP2 exhibits striking context-dependent functions across cancer types, necessitating tailored detection approaches. In acute myeloid leukemia (AML), WISP2 functions as a tumor suppressor with reduced expression and acetylation levels in bone marrow mononuclear cells from patients, correlating with poorer survival outcomes . Detection in hematological samples requires specialized preparation techniques, with immunoblotting more practical than immunohistochemistry. Contrastingly, in gastric cancer, WISP2 overexpression correlates with early TNM staging, better differentiation status, and improved survival . For gastric tissue analysis, immunohistochemistry with careful scoring (>20% stained cells considered positive) provides robust results . In breast cancer, WISP2/CCN5 knockdown promotes proliferation and progression, suggesting tumor-suppressive functions similar to AML . When comparing across cancer types, researchers must maintain consistent detection parameters while acknowledging tissue-specific optimal conditions. These divergent patterns highlight the importance of comprehensive characterization in each cancer context, combining protein expression analysis with functional studies to clarify WISP2's role. Researchers should always include appropriate tissue-specific controls and standardized quantification methods for meaningful cross-cancer comparisons.

How might emerging technologies enhance WISP2 detection and functional analysis?

Emerging technologies offer exciting opportunities to advance WISP2 research beyond current limitations. Single-cell proteomics approaches could reveal cell-type specific WISP2 expression patterns within heterogeneous tumors, providing insights into microenvironmental regulation that bulk tissue analysis cannot capture. Mass spectrometry-based techniques would allow comprehensive mapping of WISP2 post-translational modifications beyond the currently identified K6 acetylation site, potentially uncovering additional regulatory mechanisms . CRISPR-Cas9 gene editing enables precise manipulation of endogenous WISP2 and creation of specific point mutations (such as K6R) to study modification sites in their native genomic context, improving upon overexpression models . Proximity labeling methods like BioID or APEX could identify transient WISP2 interaction partners in living cells, expanding our understanding of its signaling networks. Advanced imaging techniques including super-resolution microscopy would reveal WISP2 subcellular localization with unprecedented detail. Computational approaches integrating WISP2 expression data with multi-omics datasets could identify novel regulatory relationships and predict patient responses to therapies targeting WISP2-associated pathways. These technologies collectively promise to deepen our mechanistic understanding of WISP2 biology and potentially reveal new therapeutic opportunities in cancers where WISP2 functions as a tumor suppressor.

What are the implications of WISP2 research for targeted cancer therapies?

WISP2 research has significant implications for developing novel targeted cancer therapies. The discovery that WISP2 functions as a tumor suppressor in AML suggests therapeutic strategies aimed at restoring or enhancing WISP2 expression or activity could inhibit leukemia progression . Notably, histone deacetylase inhibitors (HDACi) including valproic acid, trichostatin A, and the HDAC3-specific inhibitor RGFP966 increase WISP2 acetylation at lysine K6, preventing its degradation and enhancing its tumor-suppressive functions . This mechanism provides rationale for combining HDAC inhibitors with conventional chemotherapies in AML patients with low WISP2 expression. Additionally, targeting the ubiquitin-proteasome pathway, specifically the E3 ligase NEDD4 that mediates WISP2 degradation, represents another therapeutic avenue . In gastric cancer, where WISP2 overexpression correlates with better prognosis, therapies maintaining or enhancing WISP2 expression might improve outcomes . WISP2's role in suppressing epithelial-mesenchymal transition and matrix metalloproteinase activity suggests it could inhibit metastasis, potentially informing anti-metastatic treatment strategies . For personalized medicine approaches, stratifying patients based on WISP2 expression levels might identify those likely to benefit from specific therapeutic interventions. These translational opportunities underscore the importance of continued basic and preclinical research to fully elucidate WISP2's complex roles across cancer types.