WIF1 Antibody, Biotin conjugated

What is WIF1 and what is its biological significance?

WIF1 (Wnt inhibitory factor 1) is a secreted protein that binds to WNT proteins and inhibits their activities. It functions as an important negative regulator of the Wnt/β-catenin signaling pathway, which plays crucial roles in embryonic development, tissue homeostasis, and cancer progression. WIF1 may be involved in mesoderm segmentation during development .

Recent research has demonstrated that WIF1 functions as a tumor suppressor in various cancers. It inhibits cancer stemness and induces cellular senescence, particularly in salivary gland tumor cells. WIF1 has been shown to suppress tumor progression through upregulation of tumor-suppressor genes such as p53 and p21, while diminishing the number of cancer stem cells and inhibiting anchorage-independent cell growth .

What applications are WIF1 antibodies suitable for?

WIF1 antibodies have been validated for several research applications:

The antibody has been tested and confirmed to work with human samples, with predicted reactivity for mouse and rat samples based on sequence homology .

What is biotin conjugation and why is it advantageous for WIF1 antibody experiments?

Biotin conjugation is a chemical process that covalently attaches biotin molecules to antibodies. This modification allows the antibody to be used in conjunction with secondary reagents coupled to biotin-binding proteins such as avidin or streptavidin .

The primary advantages of using biotin-conjugated WIF1 antibody include:

• Enhanced sensitivity: The biotin-streptavidin system offers one of the strongest non-covalent biological interactions known, providing signal amplification.

• Versatility: Biotin-conjugated antibodies can be paired with various detection systems (HRP, fluorescent dyes, gold particles) coupled to streptavidin.

• Multiplex capability: Allows for simultaneous detection of multiple targets when combined with other detection methods.

• Reduced background: Can help minimize non-specific binding compared to secondary antibody methods .

What are the optimal storage conditions for biotin-conjugated WIF1 antibody?

For maximum stability and performance of biotin-conjugated WIF1 antibody, the following storage conditions are recommended:

• Store at -20°C for long-term preservation.

• Store in buffer containing PBS with 0.02% sodium azide and 50% glycerol at pH 7.3.

• Aliquoting is unnecessary for -20°C storage with glycerol-containing buffers.

• Smaller-sized aliquots (e.g., 20μl) may contain 0.1% BSA as a stabilizer.

• The conjugated antibody is typically stable for one year after shipment when stored properly .

Avoid repeated freeze-thaw cycles as they can degrade both the antibody and the biotin conjugate, potentially resulting in decreased performance in downstream applications.

How does WIF1 expression correlate with cancer progression mechanisms?

WIF1 functions as a tumor suppressor through multiple mechanisms affecting cancer stemness and progression:

• Suppression of cancer stemness markers: WIF1 re-expression in tumor cells causes significant reduction in the expression of pluripotency and stemness markers including OCT4 and c-MYC. It also downregulates adult stem cell self-renewal and multi-lineage differentiation markers such as WNT3A, TCF4, c-KIT, and MYB .

• microRNA regulation: WIF1 significantly increases the expression of pri-let-7a and pri-miR-200c, which are negative regulators of stemness and cancer progression .

• Epithelial-mesenchymal transition inhibition: WIF1 functions as a positive regulator of miR-200c, leading to downregulation of BMI1, ZEB1, and ZEB2, with a consequent increase in downstream targets such as E-cadherin .

• Cellular senescence induction: WIF1 re-expression promotes cellular senescence, possibly through upregulation of tumor-suppressor genes p53 and p21 .

When designing experiments to investigate these mechanisms, researchers should consider using biotin-conjugated WIF1 antibodies in combination with markers for cellular senescence, stemness, and EMT to establish comprehensive mechanistic insights.

What are the technical considerations for optimizing biotin-conjugated WIF1 antibody in Western blotting?

For optimal Western blot results with biotin-conjugated WIF1 antibody, consider the following technical parameters:

• Sample preparation:

  • Human fetal heart tissue lysate (20 μg) and human fetal lung tissue lysate (10 μg) have been successfully used .

  • Cell lines including Caco-2, SW480, COLO 320, and HT-29 show detectable WIF1 expression .

• Antibody dilution optimization:

  • For standard WIF1 antibody, dilutions of 1:2000-1:5000 have been effective .

  • When using the biotin-conjugated version, start with similar dilutions but optimize based on signal-to-noise ratio.

• Blocking conditions:

  • 5% non-fat dry milk (NFDM) in TBST has been validated as an effective blocking buffer .

• Detection system:

  • For biotin-conjugated antibodies, use streptavidin-HRP at an optimized dilution (typically 1:1000-1:5000).

• Band interpretation:

  • Expected molecular weight: 41-42 kDa

  • Observed molecular weight in validated studies: 41 kDa

The sample-dependent nature of WIF1 detection requires protocol optimization for specific experimental conditions, with careful attention to negative controls and validation of specificity.

How can biotin-conjugated WIF1 antibody be incorporated into multiplex imaging experiments?

Biotin-conjugated WIF1 antibody is particularly well-suited for multiplex imaging due to its compatibility with various detection systems:

• Fluorescence microscopy multiplex strategies:

  • Use streptavidin conjugated to different fluorophores (Alexa Fluor dyes, DyLight dyes) for flexible detection.

  • Can be combined with directly labeled antibodies against other targets for co-localization studies.

  • The conjugation-ready format is designed for use with fluorochromes, metal isotopes, oligonucleotides, and enzymes .

• Mass cytometry applications:

  • Biotin-conjugated WIF1 antibody can be detected using metal-tagged streptavidin in mass cytometry panels.

  • Enables simultaneous detection of WIF1 with numerous other proteins in single cells .

• Experimental design considerations:

  • Include appropriate controls for biotin background (endogenous biotin can interfere with specific binding).

  • Use biotin blocking steps when working with biotin-rich tissues.

  • Validate spectral overlap and signal spillover when designing multiplex panels.

• Signal amplification options:

  • Tyramide signal amplification can be applied with biotin-streptavidin systems for enhanced sensitivity.

  • Consider sequential detection approaches when working with multiple biotin-conjugated antibodies.

This flexibility makes biotin-conjugated WIF1 antibody valuable for complex experimental designs investigating WIF1 in relation to other Wnt pathway components or cancer markers.

How can non-specific binding be addressed when using biotin-conjugated WIF1 antibody?

When encountering non-specific binding with biotin-conjugated WIF1 antibody, implement these troubleshooting strategies:

• Endogenous biotin blocking:

  • Pre-block tissues or cells with avidin/streptavidin followed by free biotin to saturate endogenous biotin.

  • Use commercial biotin blocking kits before applying biotin-conjugated primary antibodies.

• Optimization of blocking conditions:

  • 5% NFDM in TBST has been validated for WIF1 antibody applications .

  • Alternative blocking agents such as BSA or commercial blockers may be needed for tissues with high non-specific binding.

• Antibody titration:

  • Perform careful titration experiments to determine the optimal concentration that maximizes specific signal while minimizing background.

  • For Western blot applications, WIF1 antibody dilutions from 1:1000 to 1:6000 have been effective depending on the sample .

• Validation with multiple detection methods:

  • Compare results between biotin-conjugated and unconjugated WIF1 antibody.

  • Use secondary detection methods to confirm specificity of observed signals.

• Inclusion of appropriate controls:

  • Use isotype controls conjugated to biotin to identify non-specific binding.

  • Include samples known to be negative for WIF1 expression.

What controls should be included when studying WIF1's role in cancer stemness?

• Expression controls:

  • Positive controls: Human fetal heart and lung tissue lysates have demonstrated consistent WIF1 expression patterns .

  • Negative controls: Include tissues or cell lines with validated absence of WIF1 expression.

• Functional controls for WIF1 activity:

  • WIF1 knock-down/knock-out models to demonstrate specificity of observed phenotypes.

  • WIF1 overexpression systems to validate suppressive effects on stemness markers.

• Pathway interaction controls:

  • Include assessment of canonical Wnt pathway components (β-catenin, TCF4) to confirm WIF1's mechanistic action.

  • Monitor expression of downstream targets affected by WIF1, such as OCT4, c-MYC, and miR-200c .

• Stemness marker validation:

  • Use established stemness markers alongside WIF1 detection:

    • Pluripotency markers: OCT4, c-MYC

    • Adult stem cell markers: WNT3A, TCF4, c-KIT, MYB

  • Compare results across multiple stemness assays (e.g., sphere formation, ALDH activity, side population).

• Senescence markers correlation:

  • As WIF1 induces cellular senescence, include senescence markers (SA-β-gal, p53, p21) to confirm this functional outcome .

How should researchers interpret and validate WIF1 expression patterns across different cancer types?

When analyzing WIF1 expression patterns across different cancer types, researchers should consider:

• Tissue-specific expression baseline:

  • Establish normal tissue expression levels of WIF1 for accurate comparison with tumor samples.

  • Different tissues have varying baseline WIF1 expression that must be considered when interpreting changes in cancer.

• Mechanistic context interpretation:

  • WIF1 downregulation has been observed in salivary gland carcinoma ex-pleomorphic adenoma (CaExPA) and its precursor lesion pleomorphic adenoma (PA), indicating a higher risk of malignant progression .

  • WIF1 downregulation may occur through diverse mechanisms including promoter hypermethylation and loss of heterozygosity .

• Multiple detection methods validation:

  • Compare protein detection (using biotin-conjugated and unconjugated antibodies) with mRNA expression.

  • Use immunohistochemistry to assess spatial distribution alongside Western blot for quantitative assessment.

• Functional validation experiments:

  • Re-expression of WIF1 in cancer cell lines can:

    • Inhibit cell proliferation

    • Induce a more differentiated phenotype

    • Promote cellular senescence

    • Diminish the number of cancer stem cells

    • Reduce anchorage-independent cell growth

• Clinical correlation analysis:

  • Correlate WIF1 expression levels with patient outcomes and clinicopathological features.

  • Consider WIF1 expression in the context of tumor stage, grade, and metastatic potential.

What are the optimal biotin conjugation methods for WIF1 antibody?

For optimal biotin conjugation of WIF1 antibody, researchers should consider:

• Pre-conjugation considerations:

  • The antibody should ideally be in 10-50mM amine-free buffer (e.g., MES, MOPS, HEPES, PBS) with pH 6.5-8.5 .

  • Recommended antibody concentrations of 1-4 mg/ml generally give optimal results .

• Conjugation protocol elements:

  • Add 1 μl of modifier reagent to each 10 μl of antibody solution before mixing with Biotin Mix.

  • Resuspend gently by withdrawing and re-dispensing the liquid using a pipette.

  • Allow conjugation to proceed at room temperature (20-25°C) for 3 hours in the dark. Overnight incubation is also acceptable .

• Recommended antibody quantities based on kit size:

  Kit Size	Amount of Antibody	Volume of Antibody
  3 x 10 μg Kit	10-20 μg	4-10 μl
  3 x 100 μg Kit	100-200 μg	40-100 μl
  1 x 1 mg Kit	1-2 mg	400-1000 μl

• Quality control testing:

  • After conjugation, validate the biotin-conjugated WIF1 antibody using known positive controls such as human fetal heart or lung tissue lysates.

  • Compare performance to the unconjugated antibody to ensure conjugation hasn't affected specificity or sensitivity.

How can researchers effectively use biotin-conjugated WIF1 antibody to study Wnt signaling pathway interactions?

To effectively use biotin-conjugated WIF1 antibody in Wnt signaling pathway research:

• Co-immunoprecipitation studies:

  • Use biotin-conjugated WIF1 antibody to pull down WIF1 protein complexes, followed by streptavidin capture.

  • Analyze co-precipitated Wnt proteins to identify specific interaction partners.

  • The antibody has been validated for immunoprecipitation applications at a 1:60 dilution .

• Chromatin immunoprecipitation approaches:

  • For studying WIF1's influence on gene regulation, biotin-conjugated antibodies can be used in ChIP experiments to identify genomic regions affected by WIF1-mediated Wnt inhibition.

  • Target analysis of promoter regions for stemness genes like OCT4 and c-MYC that are repressed by WIF1.

• Competitive binding assays:

  • Design experiments to assess how WIF1 competes with Frizzled receptors for Wnt ligand binding.

  • Quantify binding affinities and inhibitory effects on downstream signaling.

• Wnt reporter assays with WIF1 modulation:

  • Use TOP/FOP flash reporter systems to measure canonical Wnt pathway activity.

  • Correlate WIF1 expression (detected with biotin-conjugated antibody) with reporter activity.

  • Document changes in reporter activity with WIF1 overexpression or knockdown.

• Signaling pathway cross-talk analysis:

  • Investigate how WIF1-mediated Wnt inhibition affects other pathways (Notch, Hedgehog, TGF-β).

  • Use multiplex approaches combining biotin-conjugated WIF1 antibody with markers for these other pathways.

What methodological approaches can resolve contradictory data about WIF1 expression and function?

When facing contradictory data about WIF1 expression and function, implement these methodological approaches:

• Standardization of detection methods:

  • Use consistent antibody clones and detection systems across experiments.

  • Standardize protein loading and normalization procedures for Western blots.

  • For biotin-conjugated antibodies, ensure consistent biotin blocking protocols are applied.

• Comprehensive isoform analysis:

  • WIF1 antibodies have a predicted band size of 41 kDa and observed band size of 41 kDa in validated studies .

  • Investigate potential isoforms or post-translational modifications that might affect detection.

  • Use multiple antibodies targeting different epitopes to confirm detection consistency.

• Tissue and cellular contextualization:

  • WIF1 function may be context-dependent across different tissues and cancer types.

  • Document the cellular microenvironment and differentiation status of the samples being compared.

  • Account for heterogeneity within tumor samples that might lead to variable results.

• Methodological triangulation:

  • Combine protein detection (immunoblotting, immunohistochemistry) with mRNA analysis (qPCR, RNA-seq).

  • Correlate protein expression with functional assays measuring Wnt pathway activity.

  • Use genetic approaches (CRISPR, shRNA) to validate antibody specificity and functional observations.

• Mechanistic validation:

  • For contradictory functional data, test WIF1's effects on well-established downstream targets:

    • Stemness markers: OCT4, c-MYC, WNT3A, TCF4

    • MicroRNA regulation: pri-let-7a, pri-miR-200c

    • EMT regulators: BMI1, ZEB1, ZEB2, E-cadherin

  • Document changes in cellular phenotypes: proliferation, differentiation, senescence, anchorage-independent growth.

By employing these methodological strategies, researchers can resolve contradictions and build a more coherent understanding of WIF1's role in normal biology and cancer progression.

What emerging applications might benefit from biotin-conjugated WIF1 antibody technology?

Emerging research areas that could benefit from biotin-conjugated WIF1 antibody include:

• Single-cell proteomics:

  • Biotin-conjugated WIF1 antibody can be integrated into mass cytometry panels for high-dimensional single-cell analysis.

  • This enables correlation of WIF1 expression with dozens of other proteins at single-cell resolution.

  • The conjugation-ready format is specifically designed for these advanced flow-based assays .

• Spatial transcriptomics integration:

  • Combined protein-RNA detection methods could use biotin-conjugated WIF1 antibody alongside RNA probes.

  • This would allow correlation between WIF1 protein expression and transcriptional programs at spatial resolution.

• Liquid biopsy development:

  • Biotin-conjugated WIF1 antibody could be employed in capture systems for circulating tumor cells.

  • This approach might identify cancer cells with altered WIF1 expression as a biomarker for progression or treatment response.

• Therapeutic target validation:

  • As WIF1 suppresses cancer stemness, biotin-conjugated antibodies could help validate therapeutic approaches aimed at restoring WIF1 function.

  • This includes screening compound libraries for molecules that upregulate WIF1 expression or mimic its function.

• Extracellular vesicle characterization:

  • Biotin-conjugated WIF1 antibody could help characterize WIF1 in extracellular vesicles and analyze its role in intercellular communication in the tumor microenvironment.

How might biotin-conjugated WIF1 antibody contribute to understanding epigenetic regulation in cancer?

Biotin-conjugated WIF1 antibody can provide valuable insights into epigenetic regulation in cancer through:

• Chromatin immunoprecipitation sequencing (ChIP-seq):

  • Investigate how WIF1 expression correlates with chromatin modifications.

  • Identify transcription factors that regulate WIF1 expression through biotin-mediated pulldown approaches.

• DNA methylation correlation studies:

  • WIF1 downregulation occurs through promoter hypermethylation in salivary gland tumors .

  • Biotin-conjugated WIF1 antibody can help correlate protein expression with methylation status at single-cell resolution.

• Histone modification patterns:

  • Examine how WIF1 expression correlates with specific histone marks associated with gene activation or repression.

  • Study changes in these patterns during cancer progression and treatment response.

• microRNA regulation mechanisms:

  • WIF1 increases expression of pri-let-7a and pri-miR-200c, negative regulators of stemness and cancer progression .

  • Biotin-conjugated WIF1 antibody can help investigate protein-RNA interactions in this regulatory network.

• Therapeutic implications for epigenetic drugs:

  • Use biotin-conjugated WIF1 antibody to assess how epigenetic modifying drugs (DNA methyltransferase inhibitors, histone deacetylase inhibitors) affect WIF1 expression.

  • This approach could help identify patients who might benefit from epigenetic therapies aimed at restoring WIF1 function.

This research direction has particular relevance since epigenetic silencing of WIF1 appears to be a key mechanism contributing to its downregulation in cancer.