PROX1 Antibody, HRP conjugated

What is PROX1 and why is it significant in developmental biology?

PROX1 (Prospero homeobox 1) is a transcription factor that belongs to the Prospero homeobox family and contains a Prospero-type homeobox DNA-binding domain. It plays fundamental roles in the early development of the central nervous system by regulating gene expression and development of postmitotic, undifferentiated neurons . Additionally, PROX1 functions as a critical regulatory protein in the development of multiple organs including the heart, eye lens, liver, pancreas, and most notably, the lymphatic system . Its significance extends to various biological processes such as cell fate determination and transcriptional regulation, making it an important research target for developmental biologists and cancer researchers .

What are the key specifications of commercially available PROX1 Antibody, HRP conjugated?

The PROX1 Antibody, HRP conjugated (e.g., CSB-PA852905LB01HU) is typically stored at -20°C or -80°C to prevent repeated freeze-thaw cycles . The antibody is commonly produced in rabbit against recombinant human Prospero homeobox protein 1 (specifically amino acids 262-477) . These antibodies are purified using Protein G with purity levels exceeding 95% . The storage buffer usually contains preservatives such as 0.03% Proclin 300 and constituents including 50% Glycerol and 0.01M PBS at pH 7.4 . The direct HRP conjugation allows for streamlined detection protocols without requiring secondary antibodies, particularly beneficial in ELISA applications .

What are the recommended applications and dilutions for PROX1 antibody experimental protocols?

The PROX1 antibody demonstrates efficacy across multiple experimental applications with specific recommended dilutions:

It is strongly recommended that researchers optimize these dilutions for their specific experimental systems to obtain optimal results, as the effective concentration may be sample-dependent . Additionally, when using HRP-conjugated antibodies, researchers should consider the signal strength requirements and potential background issues when determining optimal concentrations.

How should researchers design experiments to investigate PROX1's role in transcriptional regulation of target genes?

When designing experiments to study PROX1's transcriptional regulatory functions, researchers should implement a multi-faceted approach that combines:

• Promoter binding analysis: Chromatin immunoprecipitation (ChIP) assays can be utilized to determine if PROX1 directly binds to the promoter region of target genes, as demonstrated in the regulation of MMP14 .

• Transcriptional activity assessment: Luciferase reporter assays containing the promoter region of potential target genes can determine if PROX1 activates or suppresses transcription .

• Expression correlation studies: PROX1 expression manipulation (overexpression and silencing) followed by qRT-PCR and Western blot analysis of potential target genes can establish cause-effect relationships .

• Functional validation: Phenotypic rescue experiments where PROX1-mediated effects are reversed by reintroducing the target gene can confirm the functional relationship, as demonstrated with MMP14 in invasive cell models .

This comprehensive approach has successfully established PROX1 as a transcriptional regulator of genes such as MMP14, revealing its important role in processes like cancer cell invasion and endothelial cell specification .

What controls should be included when using PROX1 antibodies in immunohistochemistry or immunofluorescence studies?

When conducting immunohistochemistry (IHC) or immunofluorescence (IF) studies with PROX1 antibodies, researchers should include several critical controls:

• Positive tissue/cell controls: Use tissues or cell lines with known PROX1 expression, such as HuH-7 or HepG2 cells, which have been validated for positive PROX1 detection .

• Negative controls: Include samples where PROX1 is known to be absent or samples where primary antibody is omitted to assess non-specific binding.

• Isotype controls: Include matched isotype antibodies (e.g., Mouse IgG2b for monoclonal antibodies) at equivalent concentrations to assess potential non-specific binding due to the antibody class .

• Genetic knockout/knockdown validation: When possible, include PROX1-depleted samples through siRNA or CRISPR approaches to confirm antibody specificity, similar to the validation performed in PROX1-depleted lymphatic vessels .

• Cross-reactivity assessment: When working with animal models, evaluate species cross-reactivity as documented in the antibody specifications (e.g., human, mouse, rat reactivity) .

These controls ensure reliable interpretation of experimental results and help troubleshoot potential technical issues.

How can PROX1 antibodies be used to investigate the role of PROX1 in lymphatic vessel development?

PROX1 antibodies are instrumental in studying lymphatic vessel development due to PROX1's critical role as a master regulator of lymphatic endothelial cell (LEC) fate. A comprehensive research approach includes:

• Lineage tracing studies: Use PROX1 antibodies in conjunction with lymphatic markers (LYVE-1, podoplanin) and blood vessel markers (PECAM, endomucin) to track the differentiation of LECs from venous endothelial cells .

• Conditional knockout models: Apply PROX1 antibodies to validate the efficiency of genetic deletion in models such as the Prox1^flox/flox; Cdh5-CreER^T2 mouse model, where 4-hydroxytamoxifen treatment induces PROX1 deletion specifically in endothelial cells .

• Marker analysis: Combine PROX1 immunostaining with other lymphatic markers to assess the impact of PROX1 manipulation on lymphatic vessel identity, as demonstrated in studies where PROX1 depletion affected expression of lymphatic markers like VEGFR-3 and NRP2 .

• Functional studies: Correlate PROX1 expression patterns with functional parameters of lymphatic vessels, such as permeability, contractility, and valve formation.

This multifaceted approach has revealed that PROX1 not only initiates lymphatic differentiation but also maintains lymphatic identity through various mechanisms, including the regulation of target genes such as MMP14 .

What experimental designs can elucidate PROX1's context-dependent roles in cancer progression?

PROX1 exhibits context-dependent functions in cancer, acting as both an oncogene and a tumor suppressor depending on the cancer type. To investigate these dual roles, researchers should design:

• Expression correlation studies: Use PROX1 antibodies for immunohistochemical analysis of patient tumor samples to correlate PROX1 expression levels with clinical outcomes, metastasis, and survival across different cancer types.

• 3D invasion models: Implement three-dimensional invasion assays using fibrin matrices to assess how PROX1 manipulation affects cancer cell invasiveness. For instance, studies have shown that PROX1 expression suppresses MMP14-dependent invasion in hepatocellular carcinoma and breast cancer models .

• Transcriptional target identification: Combine PROX1 ChIP-seq with RNA-seq after PROX1 modulation to identify cancer-type specific transcriptional targets and regulatory networks.

• Functional rescue experiments: Design experiments where PROX1-mediated phenotypes are rescued by modulating downstream targets, as demonstrated by the restoration of invasiveness in PROX1-expressing MDA-MB-231 cells after MMP14 reintroduction .

• In vivo metastasis models: Develop xenograft models with PROX1-modulated cancer cells to assess the impact on tumor growth, angiogenesis, lymphangiogenesis, and metastasis in a physiological context.

These approaches have revealed PROX1's suppressive effect on the transcription of MMP14, a metalloprotease involved in cancer invasion and angiogenesis, demonstrating one mechanism by which PROX1 can inhibit cancer progression in certain contexts .

What are the molecular mechanisms through which PROX1 regulates MMP14 expression and how can this be experimentally verified?

PROX1 negatively regulates MMP14 expression through direct interaction with its promoter. To experimentally verify this mechanism, researchers should implement:

• Promoter binding studies: Chromatin immunoprecipitation (ChIP) assays using PROX1 antibodies to demonstrate direct binding to the MMP14 promoter region .

• Promoter activity assays: Luciferase reporter assays with the MMP14 promoter to quantify the suppressive effect of PROX1 on transcriptional activity .

• Expression manipulation experiments:

  • siRNA-mediated PROX1 silencing in PROX1-positive cells (e.g., LECs, HEK293FT) followed by qRT-PCR and Western blotting to demonstrate increased MMP14 expression .

  • Ectopic expression of wild-type PROX1 (PROX1 WT) versus DNA-binding mutant PROX1 (PROX1 MUT) to confirm the requirement of PROX1's DNA-binding capacity for MMP14 suppression .

• Functional validation: 3D invasion assays in fibrin matrices to demonstrate that:

  • PROX1 silencing increases MMP14-dependent invasion in cells like HEPG2 .

  • Ectopic PROX1 expression reduces MMP14-dependent invasion in breast cancer cells and angiogenic sprouting in blood endothelial cells .

  • MMP14 reintroduction rescues the invasive phenotype suppressed by PROX1 expression .

These experiments have established that PROX1 functions as a transcriptional repressor of MMP14, directly impacting cellular processes such as cancer invasion and angiogenesis .

What are common challenges when using HRP-conjugated PROX1 antibodies and how can they be addressed?

When working with HRP-conjugated PROX1 antibodies, researchers may encounter several technical challenges:

• High background signal: This can result from non-specific binding or excessive antibody concentration.

  • Solution: Optimize blocking conditions using 3-5% BSA or milk proteins , increase washing steps, and titrate antibody concentration to determine optimal dilution for each experimental system .

• Weak or no signal: This may occur due to insufficient antigen, degraded antibody, or suboptimal detection conditions.

  • Solution: Ensure proper sample preparation, verify antigen expression in positive control samples (e.g., HepG2 or HuH-7 cells) , and optimize incubation times and temperatures.

• Non-specific bands in Western blot: These may appear due to cross-reactivity or sample degradation.

  • Solution: Use freshly prepared samples, optimize blocking and washing conditions, and compare results with positive controls showing the expected 90 kDa band for PROX1 .

• Signal fading: HRP activity can diminish during long-term storage.

  • Solution: Store antibody at -20°C or -80°C, avoid repeated freeze-thaw cycles, and protect from light exposure as indicated in storage recommendations .

• Inconsistent results between experiments: This may be due to variability in experimental conditions or antibody lots.

  • Solution: Standardize protocols, include consistent positive and negative controls, and document antibody lot numbers for reproducibility.

Proper optimization of these parameters will significantly improve the reliability and specificity of results when using HRP-conjugated PROX1 antibodies.

How can researchers validate the specificity of PROX1 antibodies in their experimental systems?

Validating antibody specificity is crucial for generating reliable research data. For PROX1 antibodies, researchers should implement the following validation strategies:

• Genetic approaches:

  • siRNA or shRNA-mediated knockdown of PROX1 should result in reduced or absent antibody signal .

  • Conditional knockout models (e.g., Prox1^flox/flox; Cdh5-CreER^T2) provide in vivo validation systems where PROX1 expression can be temporally controlled .

• Overexpression studies:

  • Ectopic expression of tagged PROX1 in cell lines with low endogenous expression allows confirmation of antibody specificity through co-localization studies .

• Multiple antibody comparison:

  • Testing different PROX1 antibodies targeting distinct epitopes should yield consistent results in positive samples.

• Known expression pattern validation:

  • Compare antibody staining patterns with established PROX1 expression in tissues such as lymphatic vessels, liver, and neural tissues .

• Western blot analysis:

  • Confirm detection of a band at the expected molecular weight of approximately 90 kDa, accounting for post-translational modifications .

• Cross-species reactivity assessment:

  • Test antibody performance across species using samples from documented reactive species (human, mouse, rat) and compare with species-specific positive controls.

These comprehensive validation approaches ensure that experimental findings accurately reflect PROX1 biology rather than artifacts of non-specific antibody binding.

What factors should be considered when designing flow cytometry experiments using PROX1 antibodies?

When designing flow cytometry experiments with PROX1 antibodies, researchers should consider several critical factors:

• Intracellular staining protocol optimization:

  • PROX1 is a nuclear transcription factor requiring effective cell permeabilization.

  • Use appropriate fixation (e.g., 4% paraformaldehyde) followed by permeabilization (e.g., 0.1-0.5% Triton X-100 or commercial permeabilization buffers).

  • Optimize fixation and permeabilization times to maintain cellular integrity while enabling antibody access to nuclear antigens.

• Antibody concentration and incubation conditions:

  • For intracellular flow cytometry, use the recommended concentration of 0.40 μg per 10^6 cells in a 100 μl suspension .

  • Extend incubation times (typically 30-60 minutes) to ensure adequate antibody penetration into the nucleus.

• Appropriate controls:

  • Include isotype controls (Mouse IgG2b for monoclonal antibodies) to establish gating strategies.

  • Use known PROX1-positive cell lines such as HepG2 as positive controls .

  • Include PROX1-negative cell populations as negative controls.

• Multiparameter analysis design:

  • Combine PROX1 staining with cell surface markers to identify specific cell populations.

  • When studying lymphatic endothelial cells, co-stain with markers such as podoplanin or LYVE-1 .

  • Ensure proper compensation when using multiple fluorophores to account for spectral overlap.

• Signal optimization for HRP-conjugated antibodies:

  • For HRP-conjugated antibodies, ensure compatibility with flow cytometry applications.

  • Consider using primary PROX1 antibodies with fluorophore-conjugated secondary antibodies as an alternative if HRP conjugates show suboptimal performance.

Careful consideration of these factors will enhance the reliability and interpretability of flow cytometry data when analyzing PROX1 expression.

How can PROX1 antibodies be utilized to investigate the role of PROX1 in the regulation of circadian rhythm?

PROX1 has been implicated in the regulation of circadian rhythm through its repression of retinoid-related orphan receptors (RORs) and their target genes . To investigate this emerging role, researchers can design experiments using PROX1 antibodies as follows:

• Temporal expression analysis:

  • Use PROX1 antibodies for immunoblotting or immunohistochemistry to track PROX1 expression over 24-hour cycles in circadian rhythm-relevant tissues (e.g., suprachiasmatic nucleus, liver).

  • Correlate PROX1 expression patterns with core clock components such as BMAL1, NPAS2, and CRY1 .

• Chromatin occupancy studies:

  • Implement ChIP-seq with PROX1 antibodies at different time points to map temporal dynamics of PROX1 binding to promoters of clock genes.

  • Focus on RORA/G-target genes and analyze binding site overlap with ROR response elements .

• Protein-protein interaction analysis:

  • Use co-immunoprecipitation with PROX1 antibodies to identify interaction partners within the circadian clock machinery.

  • Validate interactions with RORs and other clock components using reciprocal immunoprecipitation.

• Genetic manipulation combined with circadian phenotyping:

  • After PROX1 knockdown or overexpression, use antibodies to confirm manipulation efficiency and analyze effects on expression patterns of clock genes.

  • Correlate molecular changes with behavioral or physiological circadian outputs.

• Single-cell analysis:

  • Apply PROX1 antibodies in single-cell immunofluorescence or flow cytometry to analyze cell-to-cell variability in PROX1 expression within clock-relevant tissues.

  • Correlate with single-cell transcriptomics to identify cell-specific PROX1-regulated clock networks.

This research direction could enhance our understanding of how developmental transcription factors like PROX1 contribute to the regulation of circadian rhythms and metabolism.

What novel applications are emerging for PROX1 antibodies in cancer research and therapeutic development?

Several innovative applications of PROX1 antibodies are emerging in cancer research and potential therapeutic development:

• Prognostic biomarker development:

  • PROX1 antibodies are being used to develop immunohistochemical scoring systems for patient stratification, particularly in cancers where PROX1 shows context-dependent roles .

  • Quantitative image analysis of PROX1 immunostaining could provide objective prognostic indicators.

• Therapeutic target identification:

  • PROX1 antibodies can help identify downstream targets like MMP14 that might be more druggable than transcription factors .

  • Screening for compounds that modulate PROX1 expression or activity can be validated using these antibodies.

• Precision medicine approaches:

  • PROX1 expression patterns determined by immunohistochemistry could guide treatment decisions based on the context-dependent role of PROX1 in specific cancer types.

  • Combination of PROX1 with other biomarkers might improve predictive value for therapeutic response.

• Cancer stem cell research:

  • PROX1 antibodies can help investigate the role of PROX1 in cancer stem cell populations, particularly in hepatocellular carcinoma where PROX1 is highly expressed .

  • Flow cytometry applications using PROX1 antibodies could isolate and characterize PROX1-positive cancer stem cells.

• Monitoring therapy response:

  • Serial biopsies analyzed with PROX1 antibodies could monitor changes in PROX1 expression as a marker of treatment efficacy or resistance development.

• Targeted drug delivery systems:

  • Developing nanoparticles or antibody-drug conjugates that target PROX1-expressing cells could provide novel therapeutic approaches for cancers with PROX1 overexpression.

These emerging applications highlight the expanding role of PROX1 antibodies beyond basic research into translational and clinical applications.

What are the current limitations of available PROX1 antibodies and how might future developments address these challenges?

Current PROX1 antibodies face several limitations that impact their research applications:

• Epitope specificity and isoform detection:

  • Most antibodies target specific regions of PROX1 and may not detect all potential isoforms or post-translationally modified variants.

  • Future developments should focus on generating antibodies against conserved regions to ensure comprehensive detection of all PROX1 variants.

• Cross-reactivity limitations:

  • While some antibodies show reactivity across human, mouse, and rat samples , broader cross-species reactivity would benefit comparative and evolutionary studies.

  • Next-generation antibodies should be developed against highly conserved epitopes to expand cross-species applications.

• Sensitivity in low-expression contexts:

  • Current antibodies may have insufficient sensitivity for detecting low PROX1 expression levels in certain tissues or conditions.

  • Signal amplification technologies and higher-affinity antibodies could address this limitation.

• Functional blocking capacity:

  • Most available antibodies are suitable for detection but not for functional blocking of PROX1 activity.

  • Development of antibodies that can specifically interfere with PROX1 DNA binding or protein-protein interactions would enable more sophisticated functional studies.

• Compatibility across techniques:

  • Some antibodies perform well in certain applications (e.g., Western blot) but poorly in others (e.g., ChIP).

  • Future antibodies should undergo more comprehensive validation across multiple techniques to ensure broader applicability.

Addressing these limitations through innovative antibody engineering, comprehensive validation, and application-specific optimization will significantly advance PROX1 research across multiple fields.

How can integrating PROX1 antibody-based studies with emerging technologies advance our understanding of PROX1 biology?

The integration of PROX1 antibody-based approaches with cutting-edge technologies offers exciting opportunities to deepen our understanding of PROX1 biology:

• Single-cell technologies:

  • Combining PROX1 antibodies with single-cell transcriptomics can reveal cell-specific roles of PROX1 in heterogeneous tissues.

  • Single-cell CyTOF or spectral flow cytometry using PROX1 antibodies alongside numerous other markers can elucidate PROX1's relationship with complex cellular phenotypes.

• Spatial transcriptomics and proteomics:

  • Integrating PROX1 immunohistochemistry with spatial transcriptomics can contextualize PROX1 function within tissue microenvironments.

  • Multiplexed ion beam imaging or cyclic immunofluorescence with PROX1 antibodies can map PROX1 expression relative to dozens of other proteins at subcellular resolution.

• CRISPR-based functional genomics:

  • CRISPR screens followed by PROX1 antibody-based readouts can identify regulators of PROX1 expression or function.

  • CRISPR-mediated tagging of endogenous PROX1 combined with antibody detection can enable live-cell imaging of PROX1 dynamics.

• Organoid and patient-derived models:

  • PROX1 antibodies can track developmental processes and disease progression in 3D organoid cultures that better recapitulate in vivo conditions.

  • Patient-derived xenografts analyzed with PROX1 antibodies can connect PROX1 expression patterns to clinical outcomes.

• High-throughput drug screening:

  • Automated immunofluorescence using PROX1 antibodies can screen compound libraries for modulators of PROX1 expression or localization.

  • Combining such screens with functional readouts could identify therapeutic approaches for PROX1-related pathologies.

• Machine learning integration:

  • Applying machine learning algorithms to large datasets of PROX1 immunostaining patterns could identify subtle expression patterns associated with disease outcomes.

  • AI-powered image analysis could standardize and objectify PROX1 expression quantification across research and clinical settings.