ZKSCAN5 Antibody, Biotin conjugated

What is ZKSCAN5 and why is it relevant to cancer research?

ZKSCAN5 is a transcription factor containing zinc finger domains along with KRAB (Krüppel-associated box) and SCAN domains. It has gained significant research interest due to its role in cancer progression, particularly in breast cancer. ZKSCAN5 functions by activating VEGFC (Vascular Endothelial Growth Factor C) expression through recruitment of the histone methyltransferase SETD7 to the VEGFC promoter region . This activation enhances lymphangiogenesis (growth of lymphatic vessels), which plays a crucial role in cancer metastasis and immune response modulation. Research has shown that ZKSCAN5 expression is frequently upregulated in breast cancer patients and correlates positively with VEGFC expression and the number of lymphatic microvessels, making it a potential therapeutic target and prognostic marker .

What are the fundamental characteristics of biotin-conjugated antibodies?

Biotin-conjugated antibodies contain biotin molecules (vitamin H) covalently attached to the antibody structure. This conjugation provides significant advantages in research applications due to biotin's extremely high affinity for avidin and streptavidin proteins (Kd ≈ 10^-15 M). The biotin-streptavidin interaction is one of the strongest non-covalent biological interactions known, making it highly specific and stable under various experimental conditions. Biotin-conjugated antibodies typically maintain their binding specificity while gaining versatility through the biotin tag, allowing for multiple detection methods and signal amplification strategies. These antibodies are typically stored in buffered solutions containing stabilizers such as BSA (Bovine Serum Albumin) and glycerol, and properly stored at -20°C can maintain their reactivity for approximately 12 months .

What are the primary applications for ZKSCAN5 antibodies in research?

ZKSCAN5 antibodies are utilized in multiple research applications, with the most common being Western Blotting (WB), Immunohistochemistry (IHC), Immunofluorescence (IF), and Enzyme-Linked Immunosorbent Assay (ELISA) . These applications enable researchers to:

• Detect and quantify ZKSCAN5 protein expression in tissue samples and cell lines

• Investigate ZKSCAN5's subcellular localization

• Study protein-protein interactions involving ZKSCAN5

• Examine the role of ZKSCAN5 in transcriptional regulation

• Analyze ZKSCAN5's involvement in cancer progression mechanisms

For biotin-conjugated versions specifically, the applications extend to include streptavidin-based detection systems, which offer enhanced sensitivity and versatility in experimental protocols .

How do I determine the appropriate dilution for ZKSCAN5 antibody applications?

The optimal dilution for ZKSCAN5 antibodies varies by application and specific antibody characteristics. Based on available antibody data, recommended dilution ranges are:

• Western Blotting: 1:300-5000

• ELISA: 1:500-1000

• Immunohistochemistry: Typically 1:100-500

For biotin-conjugated antibodies, these ranges remain applicable, though optimization is recommended for each specific experimental setup . Optimization should include:

• Testing a dilution series (e.g., 1:100, 1:500, 1:1000)

• Including appropriate positive and negative controls

• Evaluating signal-to-noise ratio across dilutions

• Considering the abundance of your target protein in your specific samples

• Adjusting incubation time and temperature based on signal strength

How does biotin conjugation affect ZKSCAN5 antibody performance in ChIP experiments?

For Chromatin Immunoprecipitation (ChIP) experiments investigating ZKSCAN5's interaction with the VEGFC promoter, biotin-conjugated antibodies offer several advantages. ZKSCAN5 has been shown to occupy the -658 to -608 bp region of the VEGFC promoter, where it recruits SETD7 to form a transcriptionally active complex . When using biotin-conjugated ZKSCAN5 antibodies for ChIP:

• The streptavidin-biotin interaction provides stronger and more specific pulldown compared to protein A/G-based methods

• Sequential ChIP (Re-ChIP) protocols benefit from the biotin tag by allowing more efficient elution and reprecipitation

• The signal-to-noise ratio typically improves due to reduced non-specific binding

• Washing conditions can be more stringent without losing target proteins

What methodological considerations are important when using biotin-conjugated ZKSCAN5 antibodies in cancer tissue analysis?

When analyzing ZKSCAN5 expression in cancer tissues using biotin-conjugated antibodies, several methodological considerations are critical:

• Endogenous biotin blocking: Cancer tissues often contain high levels of endogenous biotin, which can lead to false-positive signals. Pre-treatment with avidin/biotin blocking kits is essential.

• Fixation method impact: Different fixation methods affect epitope accessibility. For ZKSCAN5 detection, 10% neutral-buffered formalin fixation followed by appropriate antigen retrieval (typically heat-induced epitope retrieval at pH 6.0) yields optimal results.

• Detection system selection: When using biotin-conjugated primary antibodies, avoid ABC (Avidin-Biotin Complex) detection systems to prevent cross-reactivity. Instead, use streptavidin conjugated directly to enzymes or fluorophores.

• Correlation with VEGFC expression: Since ZKSCAN5 regulates VEGFC expression, dual staining protocols to visualize both proteins simultaneously can provide valuable insights into their spatial relationship in tumor tissues .

• Quantification approaches: For meaningful correlation with clinical outcomes, standardized scoring methods should be established based on:

  • Staining intensity (0-3+)

  • Percentage of positive cells

  • Subcellular localization (nuclear vs. cytoplasmic)

How do I optimize immunoprecipitation protocols for studying ZKSCAN5 protein complexes using biotin-conjugated antibodies?

Optimizing immunoprecipitation (IP) protocols for ZKSCAN5 complexes requires careful consideration of several factors, particularly when investigating its interactions with proteins like SETD7:

• Lysis buffer composition:

  • For ZKSCAN5-SETD7 interactions, use lysis buffers containing:

    • 50 mM Tris-HCl (pH 7.5)

    • 150 mM NaCl

    • 1% NP-40 or 0.5% Triton X-100

    • 1 mM EDTA

    • Protease inhibitor cocktail

  • Avoid harsh ionic detergents that may disrupt protein-protein interactions

• Cross-linking considerations:

  • For transient ZKSCAN5 interactions, consider using cell-permeable cross-linkers (1-2 mM DSP or 1% formaldehyde) before lysis

  • Cross-linking should be optimized as excessive cross-linking may mask antibody epitopes

• Streptavidin bead selection:

  • For biotin-conjugated antibodies, magnetic streptavidin beads offer better recovery and lower background

  • Pre-clear lysates with unconjugated beads to reduce non-specific binding

• Elution strategies:

  • Competitive elution with biotin (2-5 mM) preserves protein complexes for downstream analysis

  • For complete protein recovery, boiling in SDS sample buffer is more effective but disrupts protein-protein interactions

• Validation approaches:

  • Confirm specificity through reciprocal co-immunoprecipitation as demonstrated for ZKSCAN5-SETD7 interactions

  • Include appropriate negative controls (IgG matching the host species of the primary antibody)

What are the challenges and solutions for using biotin-conjugated antibodies in multiplex immunofluorescence studies of ZKSCAN5?

For optimal results in ZKSCAN5 multiplex studies, consider using the biotin-conjugated antibody to detect ZKSCAN5 while using directly conjugated antibodies for other targets like VEGFC or lymphatic endothelial markers.

How can biotin-conjugated ZKSCAN5 antibodies be utilized to study lymphangiogenesis in cancer models?

Research has established ZKSCAN5's role in promoting lymphangiogenesis through VEGFC regulation, making it relevant for investigating cancer metastasis mechanisms . Biotin-conjugated ZKSCAN5 antibodies can be employed in multiple methodological approaches:

• Ex vivo lymphatic vessel visualization:

  • Whole-mount staining of resected tumors using biotin-conjugated ZKSCAN5 antibodies coupled with lymphatic endothelial cell markers (LYVE-1, podoplanin)

  • Streptavidin-fluorophore detection provides enhanced sensitivity for co-localization analysis

  • 3D reconstruction of confocal z-stacks to analyze lymphatic vessel density and morphology

• In vitro tube formation assay analysis:

  • Assessment of conditioned media from ZKSCAN5-manipulated cancer cells on human lymphatic endothelial cell (HLEC) tube formation

  • Quantification of key parameters including tube length, branch points, and loop formation

  • This approach has confirmed that conditioned medium from ZKSCAN5 knockdown breast cancer cells constrains tube formation, an effect rescued by ZKSCAN5 re-expression

• In vivo lymphangiogenesis monitoring:

  • Implantation of tumor cells with modified ZKSCAN5 expression followed by intra-vital imaging

  • Quantification of peritumoral and intratumoral lymphatic vessel density

  • Correlation with tumor growth and metastatic potential

  • This approach has shown that ZKSCAN5 knockdown significantly inhibits tumor growth in mouse models

• Mechanistic pathway analysis:

  • ChIP-seq using biotin-conjugated ZKSCAN5 antibodies to identify genome-wide binding sites

  • Integration with transcriptomic data to build comprehensive regulatory networks

  • Focus on VEGFC and other lymphangiogenic factors to establish a complete molecular signature

What experimental controls are essential when using biotin-conjugated ZKSCAN5 antibodies in epigenetic studies?

When investigating ZKSCAN5's role in epigenetic regulation, particularly its cooperation with histone methyltransferase SETD7 , several critical controls must be implemented:

• Input control:

  • Reserve 5-10% of chromatin before immunoprecipitation

  • Essential for normalization and determining enrichment

• Antibody specificity controls:

  • ZKSCAN5 knockdown/knockout samples

  • IgG from the same species as the primary antibody

  • Non-biotinylated version of the same antibody for comparison

• Target site controls:

  • Known ZKSCAN5 binding regions (e.g., VEGFC promoter -658 to -608 bp region)

  • Non-target regions lacking ZKSCAN5 binding sites

  • Positive control regions for associated factors (e.g., SETD7 binding sites)

• Enzymatic inhibition controls:

  • SETD7 inhibitor controls (e.g., (R)-PFI-2)

  • These have been shown to affect SETD7 recruitment to the VEGFC promoter

• Sequential ChIP validation:

  • Re-ChIP using SETD7 antibodies after ZKSCAN5 pulldown

  • Confirms co-occupancy rather than binding to adjacent sites

  • Has been verified for ZKSCAN5 and SETD7 on the VEGFC promoter

• Biotin blocking controls:

  • Pre-incubation of chromatin with free biotin

  • Ensures signals are not due to endogenous biotinylated proteins

How do I troubleshoot non-specific binding issues with biotin-conjugated ZKSCAN5 antibodies?

Non-specific binding is a common challenge when working with biotin-conjugated antibodies, including those targeting ZKSCAN5. The following troubleshooting approach addresses specific issues and solutions:

• High background in Western blots:

  • Increase blocking time using 5% BSA (preferred over milk for biotin-conjugated antibodies)

  • Add 0.1-0.5% Tween-20 to washing buffers

  • Increase washing duration and frequency (5×5 minutes)

  • Use avidin/biotin blocking kit before antibody incubation

  • Reduce primary antibody concentration (consider testing 1:1000-1:5000 dilutions)

• Multiple bands in immunoblotting:

  • Verify ZKSCAN5 expression in your cell line or tissue (predicted molecular weight: ~90 kDa)

  • Consider different extraction methods to ensure complete protein solubilization

  • Validate with alternative ZKSCAN5 antibodies to confirm band pattern

  • Use ZKSCAN5 knockdown/knockout controls to identify specific bands

• Non-specific staining in IHC/IF:

  • Implement dual blocking with both standard blocking buffer and avidin/biotin blocking system

  • Optimize antigen retrieval conditions

  • Include absorption controls (pre-incubate antibody with recombinant ZKSCAN5)

  • Use tissue from ZKSCAN5 knockdown models as negative controls

• Cross-reactivity in immunoprecipitation:

  • Pre-clear lysates with streptavidin beads before adding biotin-conjugated antibody

  • Use more stringent washing buffers (increase salt concentration to 250-300 mM)

  • Consider crosslinking antibody to streptavidin beads before immunoprecipitation

  • Validate results with alternative precipitation methods

• Signal interference in multiplexing:

  • Use spectral unmixing to distinguish between fluorescent signals

  • Apply antibodies sequentially rather than simultaneously

  • Consider tyramide signal amplification for the detection of low-abundance targets without increasing antibody concentration

What methodological approach is optimal for quantifying ZKSCAN5-mediated effects on lymphangiogenesis?

For comprehensive analysis of ZKSCAN5's impact on lymphangiogenesis, a multi-faceted methodological approach yields the most complete data:

• In vitro tube formation quantification:

  • Culture human lymphatic endothelial cells (HLECs) on Matrigel with conditioned media from ZKSCAN5-manipulated cancer cells

  • Image at 4-6 hour intervals for 24 hours

  • Quantify using automated image analysis software measuring:

    • Total tube length

    • Number of branch points

    • Number of loops/meshes

    • Average tube thickness

  • This approach has demonstrated that ZKSCAN5 knockdown impairs HLEC tube formation through reduced VEGFC secretion

• Migration and proliferation assessment:

  • Perform wound healing assays with HLECs exposed to conditioned media

  • Conduct proliferation assays (MTT, EdU incorporation, or colony formation)

  • These methods have shown that conditioned medium from ZKSCAN5 knockdown breast cancer cells decreases HLEC proliferation and migration

• In vivo lymphatic vessel density analysis:

  • Implant cancer cells with modified ZKSCAN5 expression orthotopically

  • Harvest tumors at defined timepoints

  • Perform immunohistochemistry using lymphatic vessel markers (LYVE-1, podoplanin)

  • Quantify:

    • Peritumoral lymphatic vessel density

    • Intratumoral lymphatic penetration

    • Lymphatic vessel size and morphology

    • Presence of tumor cells within lymphatic vessels

• Molecular pathway verification:

  • Measure VEGFC secretion using ELISA

  • Analyze VEGFC mRNA expression via qRT-PCR

  • Perform ChIP to confirm ZKSCAN5 binding to the VEGFC promoter

  • Assess histone modifications (particularly H3K4me1, associated with SETD7 activity) at the VEGFC promoter

How does ZKSCAN5 protein structure influence antibody selection for different applications?

ZKSCAN5 contains distinct functional domains that influence antibody selection for specific research applications:

For research focusing on ZKSCAN5's interaction with SETD7, antibodies targeting the central portion of the protein are optimal, as mapping studies have shown this region mediates the interaction with SETD7 . The studies indicate that ZKSCAN5 deletion mutants lacking specific domains showed differential binding to SETD7, with the central portion being most critical for this interaction .

When selecting biotin-conjugated ZKSCAN5 antibodies, consider:

• The epitope location relative to functional domains

• Whether the application requires preservation of protein-protein interactions

• If DNA binding capacity needs to be maintained

• The accessibility of the epitope in fixed versus native conditions

What molecular mechanisms explain ZKSCAN5's regulation of VEGFC expression, and how can antibodies help elucidate them?

ZKSCAN5 regulates VEGFC expression through a specific molecular mechanism that can be investigated using appropriately selected antibodies:

• Promoter binding and recruitment mechanism:

  • ZKSCAN5 directly binds to the VEGFC promoter region (-658 to -608 bp)

  • It recruits histone methyltransferase SETD7 to this region

  • SETD7 catalyzes H3K4 monomethylation, an activating histone modification

  • This epigenetic change promotes VEGFC transcription

• Antibody-based investigation approaches:

  • ChIP using biotin-conjugated ZKSCAN5 antibodies can confirm binding to the VEGFC promoter

  • Sequential ChIP (Re-ChIP) using ZKSCAN5 and SETD7 antibodies verifies co-occupancy

  • ChIP for H3K4me1 after ZKSCAN5 knockdown demonstrates functional impact on histone modification

  • Protein complex immunoprecipitation identifies additional cofactors in the regulatory complex

• Validation strategies:

  • ZKSCAN5 or SETD7 knockdown reduces occupancy at the VEGFC promoter

  • SETD7 inhibitors (e.g., (R)-PFI-2) prevent VEGFC upregulation despite ZKSCAN5 presence

  • Direct interaction between ZKSCAN5 and SETD7 confirmed by reciprocal co-immunoprecipitation and GST pull-down experiments

• Regulatory implications:

  • This mechanism suggests ZKSCAN5 as a potential therapeutic target to inhibit tumor lymphangiogenesis

  • Biotin-conjugated antibodies provide tools for high-throughput screening of compounds disrupting the ZKSCAN5-SETD7 interaction

What are the emerging applications of biotin-conjugated ZKSCAN5 antibodies in cancer research?

Biotin-conjugated ZKSCAN5 antibodies are finding expanding applications in cancer research, particularly given ZKSCAN5's emerging role as a prognostic factor in breast cancer . Current and developing applications include:

• Biomarker development:

  • Tissue microarray screening of large patient cohorts

  • Correlation of ZKSCAN5 expression with clinical outcomes

  • Development of standardized scoring systems for prognostic application

  • Integration into multi-marker panels for improved predictive power

• Therapeutic target validation:

  • High-throughput screening for compounds disrupting ZKSCAN5-SETD7 interaction

  • In vivo imaging of ZKSCAN5 expression response to experimental therapies

  • Target engagement studies for developing ZKSCAN5 inhibitors

  • Combination therapy approaches targeting ZKSCAN5 and VEGFC pathways

• Single-cell applications:

  • Single-cell protein profiling in heterogeneous tumor populations

  • Spatial transcriptomics integrated with ZKSCAN5 protein detection

  • Analysis of rare cell populations within the tumor microenvironment

  • Correlation of ZKSCAN5 expression with cancer stem cell markers

• Liquid biopsy development:

  • Detection of circulating tumor cells expressing ZKSCAN5

  • Correlation with metastatic potential and treatment response

  • Longitudinal monitoring of ZKSCAN5-expressing circulating tumor cells during therapy

These emerging applications build upon the established role of ZKSCAN5 in promoting lymphangiogenesis through VEGFC upregulation and its correlation with poor prognosis in breast cancer patients .

How might advances in antibody technology enhance ZKSCAN5 research in the future?

Several technological advances in antibody development are poised to enhance ZKSCAN5 research:

• Site-specific biotin conjugation:

  • Enzymatic approaches for controlled biotin positioning

  • Maintains native antibody structure and function

  • Improves batch-to-batch consistency

  • Enhances sensitivity in detection applications

• Bifunctional antibody conjugates:

  • ZKSCAN5 antibodies conjugated with both biotin and photoactivatable crosslinkers

  • Enables precise spatial control of protein complex capture

  • Facilitates identification of transient interaction partners

  • Particularly valuable for mapping ZKSCAN5's protein interaction network beyond SETD7

• Nanobody and single-domain antibody approaches:

  • Smaller antibody formats with improved tissue penetration

  • Enhanced access to epitopes in compact chromatin

  • Compatible with super-resolution microscopy techniques

  • Facilitates in vivo imaging of ZKSCAN5 expression

• Intracellular antibody delivery systems:

  • Nanoparticle-based delivery of function-blocking antibodies

  • Cell-penetrating peptide conjugated antibodies

  • Enables functional studies without genetic manipulation

  • Potential therapeutic applications targeting ZKSCAN5 function

These technological advances will expand our understanding of ZKSCAN5's role in cancer progression and potentially lead to novel therapeutic approaches targeting the ZKSCAN5-VEGFC regulatory axis.

ZKSCAN5 Detection Methods Comparison