WISP2 Antibody，Biotin conjugated

What is WISP2 and why is it an important research target?

WISP2 (WNT1 inducible signaling pathway protein 2) belongs to the connective tissue growth factor (CTGF) family. Unlike other family members, WISP2 lacks the C-terminal cystine knot-like (CT) domain implicated in dimerization and heparin binding . It functions as a matricellular protein with several significant biological roles:

• Acts as an endogenous inhibitor of collagen linearization

• Modulates bone turnover and promotes osteoblast cell adhesion

• Inhibits fibrinogen binding to integrin receptors and osteocalcin production

• Demonstrates context-dependent roles in cancer progression (inhibitory in breast cancer, promotional in ovarian cancer)

WISP2's amino acid sequence (recombinant protein) is: MQLCPTPCTC PWPPPRCPLG VPLVLDGCGC CRVCARRLGE PCDQLHVCDA SQGLVCQPGA GPGGRGALCL LAEDDSSCEV NGRLYREGET FQPHCSIRCR CEDGGFTCVP LCSEDVRLPS WDCPHPRRVE VLGKCCPEWV CGQGGGLGTQ PLPAQGPQFS GLVSSLPPGV PCPEWSTAWG PCSTTCGLGM ATRVSNQNRF CRLETQRRLC LSRPCPPSRG RSPQNSAF .

How does biotin conjugation enhance detection systems in WISP2 research?

Biotin conjugation significantly improves detection sensitivity through several mechanisms:

• Biotin binds non-covalently to avidin and streptavidin with exceptionally high affinity (Kd ≈ 10^-15 M)

• The small biotin molecule (244 Da) minimally interferes with antibody binding

• Signal amplification occurs because multiple streptavidin molecules can bind to biotinylated antibodies

• Biotin-SP (spacer) formats extend the biotin moiety away from the antibody surface by 22.4 Å, making it more accessible to streptavidin binding sites

• Provides flexibility in detection systems - a single biotinylated antibody can be used with various streptavidin conjugates (HRP, AP, fluorophores)

This design is particularly advantageous in ELISA applications where detection sensitivity is critical for accurately measuring WISP2 levels in biological samples .

What are the recommended protocols for using WISP2 Antibody, Biotin conjugated in sandwich ELISA?

For optimal sandwich ELISA performance with WISP2 Antibody, Biotin conjugated:

Standard Protocol:

• Coat plates with non-biotinylated anti-WISP2 capture antibody (e.g., PeproTech Polyclonal Anti-Human CTGFL/WISP-2 500-P212) at 0.25-1.0 μg/mL

• Block with appropriate blocker (typically 2% BSA in PBS)

• Add samples or standards

• Add biotinylated WISP2 antibody at 0.25-1.0 μg/mL (100 μL/well)

• Add streptavidin-enzyme conjugate (HRP or AP)

• Add appropriate substrate and measure signal

Key Sensitivity Parameters:

• Detection limit: 0.2-0.4 ng/well of recombinant human CTGFL/WISP-2

• For binding assays involving WISP2-collagen interactions, use 500 nM WISP2 for coating

• When investigating WISP1-WISP2 binding, incubate proteins for 45 minutes at room temperature prior to adding to coated wells

These protocols have been validated for detecting WISP2 in serum, plasma, and tissue homogenates with minimal cross-reactivity .

How can WISP2 Antibody, Biotin conjugated be used to study WISP2's role in collagen remodeling and cancer metastasis?

WISP2's role as the first known inhibitor of collagen linearization represents a significant mechanism in cancer metastasis research. The following methodological approaches leverage biotinylated WISP2 antibodies:

Experimental Design for Studying WISP2-Collagen Interactions:

• Binding Assay Protocol:

  • Prepare collagen I solution on ice as described for scanning electron microscopy

  • Coat 96-well plates with 100 μL collagen I solution (10 min at 4°C)

  • Remove excess and incubate at 37°C for 4 hours to allow fibril formation

  • For WISP1-WISP2 binding studies, coat plates with 50 μL of 500 nM WISP2 in PBS + 0.01% Tween-20 (overnight at 4°C)

  • Block with 2% BSA in PBS to prevent non-specific binding

  • Prepare WISP1/WISP2 protein concentrations in PBS with 1% BSA

  • Incubate on rotator (45 min, room temperature)

  • Add to collagen-coated wells and incubate (2 hours, 37°C)

  • Detect with anti-WISP1 or anti-WISP2 antibodies followed by appropriate conjugates

• Visualizing Collagen Architecture Changes:

  • Scanning electron microscopy reveals that WISP2 prevents WISP1-induced collagen linearization

  • Use biotinylated WISP2 antibody to track WISP2 localization in tissue sections

  • Quantify parameters including fibril curvature ratio and density of knot-like and hairpin-like structures

Key Research Findings on WISP2 in Cancer:

• WISP2 expression is lower in most solid tumors compared to normal tissues

• Restoration of WISP2 impairs collagen linearization and prevents tumor cell invasion

• WISP2 deletion in ovarian cancer cells (ES-2 and HO8910) promotes tumor growth in xenograft models

• WISP2 deletion inhibits cell growth, clone formation, and migration of ovarian cancer cells through ERK1/2, CEBPα, and CEBPβ pathways

These methodologies provide powerful tools for investigating WISP2's therapeutic potential in normalizing collagen architecture and inhibiting metastasis.

What validation methods should be employed when using WISP2 Antibody, Biotin conjugated in experimental systems?

Rigorous validation is essential when working with WISP2 Antibody, Biotin conjugated:

Comprehensive Validation Strategy:

• Specificity Validation:

  • Western blot analysis using positive control tissues (e.g., normal human mammary epithelium)

  • Include normal gastric mucosa with PBS as primary antibody for negative control

  • Confirm detection of recombinant hCTGFL/WISP-2 at 1.5-3.0 ng/lane under both reducing and non-reducing conditions

• Antibody Performance Controls:

  • Ensure biotinylation doesn't interfere with epitope binding by comparing with non-biotinylated antibody

  • Test antibody functionality with CRISPR/Cas9 WISP2 knockout cell lines (e.g., ES-2 WISP2 KO)

  • Verify avidin/streptavidin binding efficiency using known controls

• Quantitative Validation:

  • Determine intra-assay precision (CV% <8%) and inter-assay precision

  • Establish standard curves with recombinant WISP2 protein (detection range: 0.156-10 ng/mL)

  • Verify sensitivity (typical <0.039 ng/mL for high-quality assays)

• Cross-Reactivity Assessment:

  • Test against related CCN family proteins to confirm specificity

  • Evaluate performance across species (human WISP2 shares 72% amino acid identity with mouse)

Validation Data Example:

What are the critical technical considerations for optimizing Western blot detection using WISP2 Antibody, Biotin conjugated?

Optimizing Western blot protocols for WISP2 detection requires attention to several critical parameters:

Optimization Protocol:

• Sample Preparation:

  • Lyse cells by sonication in lysis buffer at 4°C

  • Centrifuge lysates and collect supernatants

  • Determine protein concentration using BCA reagent assay

• Antibody Concentration:

  • Use WISP2 Antibody, Biotin conjugated at 0.1-0.2 μg/mL concentration

  • For detection, use streptavidin-HRP conjugates

• Gel and Transfer Conditions:

  • Run samples on 10% SDS-PAGE gel

  • Transfer onto nitrocellulose membranes (PVDF alternatives may require protocol adjustment)

• Detection System:

  • Use enhanced chemiluminescence detection systems

  • For low abundance samples, consider extended exposure times or signal amplification systems

• Controls for Validation:

  • Include recombinant hCTGFL/WISP-2 as positive control

  • Test under both reducing and non-reducing conditions

  • Normalize against housekeeping proteins (e.g., GAPDH at 1:5000 dilution)

Antibody Performance Table:

How can researchers use WISP2 Antibody, Biotin conjugated to investigate WISP1-WISP2 interactions in collagen remodeling?

WISP2's inhibitory effect on WISP1-induced collagen linearization represents an important area for metastasis research. This detailed protocol enables investigation of these interactions:

Step-by-Step Protocol:

• Interaction Analysis:

  • Coat plates with collagen I solution (prepare on ice, coat for 10 min at 4°C)

  • Incubate at 37°C for 4 hours to allow fibril formation

  • For WISP1-WISP2 binding studies, pre-mix WISP1 and WISP2 in various molar ratios (1:1, 1:3) in PBS with 1% BSA

  • Incubate protein mixtures on a rotator (45 min, room temperature)

  • Add to collagen-coated wells and incubate (2 hours, 37°C)

  • Wash with 1X wash buffer (3 times)

  • Detect bound proteins using biotinylated anti-WISP2 antibody followed by streptavidin-HRP

  • Develop with appropriate substrate and measure signal

• Binding Competition Assays:

  • To test whether WISP2 disrupts WISP1-collagen interactions, use solid-phase binding assays

  • For pre-bound WISP1-Col I or WISP2-Col I complexes, wash 3 times with wash buffer

  • Add different concentrations of WISP1 or WISP2 in PBS with 1% BSA

  • Incubate (2 hours, 37°C)

  • After washing, detect bound proteins with anti-WISP1 or anti-WISP2 antibodies

Key Research Findings:

• WISP2 inhibits WISP1's function by preventing WISP1 binding to collagen I

• At 1:3 WISP1:WISP2 molar ratio, WISP2 effectively blocks WISP1-induced collagen linearization

• WISP1 and WISP2 can directly bind to each other, potentially sequestering WISP1

• Both WISP1 and WISP2 can bind to fibrillar collagen I

• Once bound to collagen, neither protein is easily displaced by the other

These methodologies provide critical insights into how WISP2 functions as an inhibitor of collagen linearization and its potential therapeutic applications in cancer metastasis.

What are common technical challenges when working with WISP2 Antibody, Biotin conjugated and how can they be addressed?

Researchers frequently encounter several challenges when working with biotinylated antibodies for WISP2 detection:

Common Issues and Solutions:

• High Background in Immunoassays:

  • Cause: Endogenous biotin in samples or inadequate blocking

  • Solution: Pre-block with avidin/streptavidin, use biotin-free blocking reagents, increase washing steps

• Reduced Sensitivity over Storage:

  • Cause: Biotin conjugate degradation

  • Solution: Store at -20°C or -80°C, avoid repeated freeze-thaw cycles , consider adding glycerol (50%) as stabilizer

• Interference in Multiplex Assays:

  • Cause: Cross-reactivity between detection systems

  • Solution: Carefully optimize antibody concentrations, use highly purified antibody preparations (>95%, antigen affinity purified)

• Inconsistent Signal Amplification:

  • Cause: Variable streptavidin binding

  • Solution: Use standardized streptavidin reagents, optimize streptavidin-enzyme concentration

• Non-specific Binding Issues:

  • Cause: Suboptimal buffer conditions

  • Solution: Use recommended buffers (PBS with 0.01% Tween-20 for binding, 1% BSA for protein preparations)

Buffer Composition Table:

How can researchers optimize signal-to-noise ratio when using WISP2 Antibody, Biotin conjugated in complex tissue samples?

Working with complex tissue samples presents unique challenges for WISP2 detection:

Optimization Strategies:

• Sample Preparation Optimization:

  • For tissue homogenates, use standardized sonication in lysis buffer at 4°C

  • Centrifuge lysates thoroughly to remove particulates

  • Consider pre-clearing samples with protein A/G beads to reduce non-specific binding

• Antigen Retrieval for Tissue Sections:

  • Optimize antigen retrieval methods for formalin-fixed paraffin-embedded tissues

  • Test both heat-induced epitope retrieval and enzymatic methods

• Signal Amplification Selection:

  • For low abundance WISP2, utilize biotin-streptavidin systems with tyramide signal amplification

  • Consider Biotin-SP (long spacer) formats which extend biotin away from the antibody surface by 22.4 Å

• Background Reduction Techniques:

  • Implement dual blocking (protein block followed by biotin/avidin blocking)

  • Use highly purified primary antibodies (affinity purified preparations)

  • Optimize antibody concentration through titration experiments

  • Include appropriate negative controls (normal rabbit IgG at matching concentration)

• Detection System Selection:

  • For low abundance targets: streptavidin-HRP with enhanced chemiluminescence

  • For multiplexed detection: streptavidin conjugated to different fluorophores

  • For highest sensitivity: alkaline phosphatase systems with chromogenic substrates

Tissue-Specific Considerations:
Based on published research, WISP2 expression varies significantly across tissues, requiring different detection strategies:

• Normal mammary epithelium: high expression (positive control)

• Gastric tissue: moderate expression

• Skeletal muscle: detectable expression (requires IHC optimization)

• Cancer tissues: often reduced expression compared to normal tissue counterparts

What emerging applications might benefit from WISP2 Antibody, Biotin conjugated detection systems?

Recent research suggests several promising directions for WISP2 antibody applications:

• Therapeutic Monitoring:

  • Track WISP2 restoration therapies in cancer treatment

  • Monitor WISP2:WISP1 ratios as biomarkers for metastatic potential

• Single-Cell Analysis:

  • Combine with microfluidic platforms for single-cell WISP2 secretion analysis

  • Integrate with mass cytometry for comprehensive protein profiling

• In Vivo Imaging:

  • Develop in vivo imaging techniques using biotinylated WISP2 antibodies paired with streptavidin-conjugated imaging agents

  • Monitor tumor microenvironment changes during treatment

• Liquid Biopsy Applications:

  • Ultrasensitive detection of circulating WISP2 as a potential biomarker

  • Paired analysis with extracellular matrix remodeling markers

• Drug Discovery Platforms:

  • High-throughput screening for compounds that modulate WISP2 activity

  • Target engagement studies for WISP2-directed therapeutics

Research Areas Table:

How might WISP2 antibody detection methods evolve to address current limitations in cancer research?

Current research points to several technological improvements that could enhance WISP2 research:

• Enhanced Specificity Reagents:

  • Development of recombinant antibody fragments with higher specificity

  • Domain-specific WISP2 antibodies to distinguish different functional regions

• Multiplexed Detection Systems:

  • Simultaneous monitoring of WISP2 and interacting partners (WISP1, collagens)

  • Integration with spatial transcriptomics to correlate protein and gene expression

• Live-Cell Imaging Applications:

  • Real-time visualization of WISP2-collagen interactions

  • Dynamic analysis of WISP2 secretion and extracellular matrix effects

• Quantitative Structure-Function Analysis:

  • Tools to correlate WISP2 levels with collagen architecture parameters

  • Automated image analysis for fibril curvature ratio and structural features

• Microenvironment Context:

  • Methods to study WISP2 function within complex 3D tumor microenvironments

  • Integration with other matricellular protein detection systems

These advancements would help overcome current limitations in understanding WISP2's context-dependent functions in cancer and other diseases, potentially leading to novel therapeutic strategies targeting the WISP2 pathway.