PROX1 Antibody, Biotin conjugated

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

PROX1 (Prospero homeobox 1) is a transcription factor belonging to the Prospero homeobox family containing a Prospero-type homeobox DNA-binding domain. It plays fundamental roles in multiple developmental processes, particularly in central nervous system formation and lymphatic system development. In the CNS, PROX1 regulates gene expression and development of postmitotic, undifferentiated neurons . Most significantly, PROX1 serves as a master regulator for lymphatic endothelial cell development, where it is essential for proper lymphangiogenesis . Research indicates that PROX1 activity in a specific subpopulation of endothelial cells in embryonic veins is required not only to promote lymphangiogenesis but also to determine lymphatic fate . The protein has a calculated molecular weight of 83 kDa but is typically observed at approximately 90 kDa in Western blot analyses .

What are the primary applications for biotin-conjugated PROX1 antibodies?

Biotin-conjugated PROX1 antibodies serve as versatile tools across multiple experimental applications in developmental biology and cancer research. Western blotting represents the primary validated application, with a recommended working dilution of 0.1 μg/mL for optimal detection of recombinant human PROX1 . Additionally, these antibodies have been cited for successful use in immunohistochemistry of frozen tissue sections, particularly for visualization of lymphatic endothelial cells . The biotin conjugation provides significant signal amplification advantages through the strong biotin-avidin interaction, making these antibodies particularly valuable for detecting low-abundance PROX1 in complex tissue samples. For ELISA applications, biotin-conjugated PROX1 antibodies enable sensitive detection without the need for secondary antibody incubation steps, streamlining experimental workflows .

How should researchers approach sample preparation for optimal PROX1 detection?

Effective sample preparation is critical for successful PROX1 detection across various experimental platforms. For Western blot applications, researchers should prepare tissue or cell lysates in a buffer containing appropriate protease inhibitors to prevent degradation of the target protein. When working with human or rat samples, positive controls such as HuH-7 cells or rat liver tissue are recommended as they consistently express detectable levels of PROX1 . For immunohistochemical applications, antigen retrieval methods significantly impact detection sensitivity. Optimal results are achieved using TE buffer at pH 9.0, though citrate buffer at pH 6.0 provides an acceptable alternative for human lung cancer tissue samples . For immunofluorescence applications, fixation protocols should be carefully optimized, with 4% paraformaldehyde fixation for 10-15 minutes typically providing good results with HuH-7 cells . Prior to antibody application, thorough blocking is essential to minimize non-specific binding, particularly when working with biotin-conjugated antibodies in tissues with high endogenous biotin content.

What are the proper storage and handling procedures for biotin-conjugated PROX1 antibodies?

Proper storage and handling of biotin-conjugated PROX1 antibodies are essential for maintaining their functionality and extending their shelf life. For long-term storage of lyophilized antibody formulations, a temperature range of -20°C to -70°C is recommended, with stability guaranteed for up to 12 months from the date of receipt under these conditions . After reconstitution, the antibody remains stable for approximately one month when stored at 2-8°C under sterile conditions, or for up to six months when aliquoted and stored at -20°C to -70°C . It is critical to avoid repeated freeze-thaw cycles as these can lead to protein denaturation and reduced antibody activity . For reconstitution, researchers should use sterile PBS to achieve a concentration of 0.2 mg/mL . Some formulations include stabilizing proteins such as BSA, which serves as a carrier protein to help maintain antibody activity . Working solutions should be prepared fresh for each experiment to ensure consistent results and minimize potential degradation from extended storage at working dilutions.

How can researchers optimize biotin-conjugated PROX1 antibodies for dual immunofluorescence applications?

Optimizing biotin-conjugated PROX1 antibodies for dual immunofluorescence requires careful consideration of detection systems and potential cross-reactivity. Begin by selecting secondary detection reagents that minimize signal overlap—streptavidin conjugated to fluorophores with minimal spectral overlap with other fluorochromes used in your experiment. When designing dual staining protocols with biotin-conjugated PROX1 antibodies, sequential staining rather than simultaneous application often yields superior results. Start with the non-biotinylated primary antibody, complete its detection, and then apply the biotin-conjugated PROX1 antibody. This approach prevents potential cross-reaction between detection systems. For optimal signal-to-noise ratio, implement a blocking step with avidin/biotin blocking kit before applying the biotin-conjugated antibody, particularly important in tissues with high endogenous biotin like liver, kidney, and brain. When examining lymphatic vessel development, combine biotin-conjugated PROX1 detection with markers such as LYVE-1 or podoplanin to precisely identify lymphatic endothelial cells undergoing differentiation . Dilution optimization is essential—start with the manufacturer's recommended range (1:200-1:800 for immunofluorescence applications) and perform a dilution series to determine optimal concentration for your specific tissue or cell type .

What experimental approaches can distinguish between PROX1's functions in lymphatic versus neural development?

Distinguishing between PROX1's diverse developmental roles requires sophisticated experimental designs that isolate tissue-specific functions. For investigating lymphatic-specific functions, conditional knockout models utilizing lymphatic endothelial cell-specific Cre drivers (such as Prox1+/Cre) can be combined with floxed Wnt pathway components (such as Wls) to evaluate PROX1's interactions with Wnt signaling specifically in lymphatic vessels . To study neural functions separately, researchers should employ neural progenitor-specific Cre systems instead. Temporal control is crucial—PROX1 expression timing differs between neural (earlier) and lymphatic (later) development, so inducible systems can target specific developmental windows. For in vitro investigation, primary cell isolation techniques can separate lymphatic endothelial cells from neural progenitors, allowing comparative analysis of PROX1 binding partners and transcriptional targets using ChIP-seq or RNA-seq approaches. Cellular localization studies using biotin-conjugated PROX1 antibodies can reveal important differences in PROX1's nuclear distribution patterns between neural and lymphatic cells. Finally, co-immunoprecipitation experiments can identify tissue-specific protein interactions, such as PROX1's documented interaction with β-catenin and TCF7L1 in lymphatic cells , which may differ from its neural interaction network.

How can researchers investigate PROX1's interaction with the Wnt/β-catenin signaling pathway?

Investigating PROX1's interaction with the Wnt/β-catenin signaling pathway requires multifaceted experimental approaches targeting protein-protein interactions and functional outcomes. Co-immunoprecipitation experiments using biotin-conjugated PROX1 antibodies represent a powerful approach to isolate PROX1-containing protein complexes, with subsequent Western blotting for β-catenin and TCF7L1 to confirm direct interactions as suggested by previous research . Chromatin immunoprecipitation (ChIP) assays using the biotin-conjugated PROX1 antibody can identify genomic regions where PROX1 binds near Wnt-responsive elements, helping elucidate the transcriptional regulatory mechanisms. For functional studies, researchers should consider implementing reporter assays with TOP/FOP flash constructs in cells with modulated PROX1 expression to measure the impact on canonical Wnt signaling activity. Genetic interaction studies, similar to those conducted using Prox1+/Cre and Wls conditional knockouts, provide in vivo evidence of pathway interaction, particularly in lymphatic vascular development . Proximity ligation assays offer an alternative method to visualize and quantify PROX1-β-catenin interactions directly within cells or tissues, providing spatial information about where these interactions occur. Finally, researchers can employ oscillatory shear stress experiments on lymphatic endothelial cells to examine how mechanical forces regulate Wnt secretion and PROX1 activity, as mechanical factors have been shown to influence this signaling axis .

What controls should be included when validating PROX1 antibody specificity in experimental designs?

Rigorous validation of PROX1 antibody specificity requires comprehensive control strategies to ensure reliable experimental outcomes. Positive control samples with confirmed PROX1 expression should be incorporated, with HuH-7 cells and rat liver tissue representing well-documented positive controls for Western blot applications . Negative controls should include tissues or cell lines with minimal PROX1 expression, while knockout/knockdown validation using PROX1 siRNA or CRISPR-edited cell lines provides the most stringent specificity assessment. Peptide competition assays, where the antibody is pre-incubated with excess immunizing peptide before application to samples, can confirm binding specificity—signals that disappear in competition samples indicate specific binding. When working with biotin-conjugated antibodies, researchers must include controls that assess potential endogenous biotin interference, particularly in biotin-rich tissues. Cross-reactivity assessment using samples from multiple species helps determine species specificity beyond the manufacturer's tested reactivity (human and rat for many PROX1 antibodies) . Finally, validation across multiple applications (Western blot, IHC, IF) using the same antibody batch enhances confidence in antibody performance, with antibody dilution series helping identify optimal working concentrations for each application while confirming signal specificity.

What are the recommended dilutions and detection systems for biotin-conjugated PROX1 antibodies across different applications?

Optimal dilutions for biotin-conjugated PROX1 antibodies vary significantly depending on the specific application and sample type. For Western blot applications, a dilution range of 1:1000-1:6000 is typically effective, with 0.1 μg/mL representing an optimal concentration for recombinant PROX1 detection . Immunohistochemistry applications generally require more concentrated antibody solutions, with recommended dilutions ranging from 1:2000-1:8000 for paraffin-embedded tissues . Immunofluorescence and immunocytochemistry applications typically employ dilutions between 1:200-1:800 . For detection systems, biotin-conjugated antibodies pair effectively with avidin/streptavidin detection methods. Streptavidin-HRP is recommended for Western blot and immunohistochemistry applications, while streptavidin conjugated to fluorophores (Alexa Fluor 488, 555, or 647) works well for immunofluorescence detection. When designing chromogenic IHC protocols, DAB (diaminobenzidine) substrate provides excellent sensitivity when paired with streptavidin-HRP systems. For all applications, researchers should conduct preliminary titration experiments to determine the optimal antibody concentration for their specific sample type, as the recommended ranges serve as starting points that may require adjustment based on target expression levels and sample characteristics .

How should researchers troubleshoot non-specific binding issues with biotin-conjugated PROX1 antibodies?

Non-specific binding is a common challenge when working with biotin-conjugated antibodies, requiring systematic troubleshooting strategies. The most frequent source of background with biotin-conjugated antibodies is endogenous biotin interference, which can be effectively mitigated by implementing an avidin/biotin blocking step before antibody application. Increasing blocking stringency represents another key approach—extending blocking time to 1-2 hours and using 5-10% normal serum from the species of the secondary detection reagent can significantly reduce non-specific binding. Optimization of antibody concentration is essential, as excessive antibody often leads to increased background; conducting a dilution series beyond the recommended range (1:1000-1:6000 for WB, 1:2000-1:8000 for IHC) can identify the minimum effective concentration . Reducing incubation temperature from room temperature to 4°C and extending incubation time can improve specificity while maintaining sensitivity. When troubleshooting Western blot applications specifically, increasing washing stringency (more washes, longer duration, higher detergent concentration) often resolves non-specific bands. For immunohistochemistry applications, antigen retrieval optimization is critical—comparing TE buffer (pH 9.0) and citrate buffer (pH 6.0) methods can significantly impact specific signal-to-noise ratio . Finally, switching to detection methods with enzyme-based signal amplification rather than direct fluorophore conjugation may provide improved signal specificity in challenging sample types.

What strategies can improve detection sensitivity when working with low PROX1 expression samples?

Detecting PROX1 in samples with low expression levels requires specialized techniques to enhance sensitivity without compromising specificity. Signal amplification represents the primary approach, with tyramide signal amplification (TSA) offering up to 100-fold signal enhancement compared to conventional detection methods when using biotin-conjugated PROX1 antibodies. Sample enrichment techniques, such as immunoprecipitation before Western blotting, can concentrate PROX1 protein from dilute samples. For tissue sections, implementing extended primary antibody incubation (overnight at 4°C rather than 1-2 hours at room temperature) allows more complete antigen binding, particularly at lower antibody concentrations. Optimized antigen retrieval is essential—for PROX1 detection, TE buffer at pH 9.0 has demonstrated superior results compared to citrate buffer alternatives . Modern detection technologies like proximity ligation assay (PLA) can detect even single protein molecules by generating amplifiable DNA circles when antibodies bind in close proximity. For Western blot applications, highly sensitive chemiluminescent substrates with long-lasting signal emission improve detection of low-abundance proteins like PROX1. When working with fixed tissues, reducing fixation time can preserve antigenicity, though this must be balanced against structural preservation. Finally, when analyzing digital images, computational approaches such as deconvolution microscopy and background subtraction algorithms can extract meaningful signals that might otherwise be overlooked in samples with marginal PROX1 expression.

What are the recommended reconstitution and storage protocols for lyophilized biotin-conjugated PROX1 antibodies?

Proper reconstitution and storage of lyophilized biotin-conjugated PROX1 antibodies is critical for maintaining their functionality and extending their usable lifespan. For reconstitution, researchers should add sterile PBS to achieve a final concentration of 0.2 mg/mL, adding the buffer slowly while gently rotating the vial to ensure complete dissolution without creating bubbles that could denature the protein . After reconstitution, the solution should stand at room temperature for approximately 30 minutes to ensure complete rehydration before use or storage. For short-term storage (up to one month), reconstituted antibody can be kept at 2-8°C under sterile conditions . For longer-term storage, the reconstituted antibody should be divided into single-use aliquots and stored at -20°C to -70°C, where it remains stable for up to six months . It is critical to avoid repeated freeze-thaw cycles as each cycle can significantly reduce antibody activity. Some commercial preparations include stabilizing proteins such as BSA (0.1%) as carrier proteins, which help maintain antibody functionality . The original lyophilized formulation, when stored properly at -20°C to -70°C, maintains stability for up to 12 months from the receipt date . For all storage conditions, antibody vials should be protected from light exposure, which can damage the biotin conjugate and reduce signal strength in subsequent applications.

How can biotin-conjugated PROX1 antibodies be utilized to study lymphangiogenesis in developmental models?

Biotin-conjugated PROX1 antibodies offer significant advantages for studying lymphangiogenesis in developmental models through various experimental approaches. Whole-mount immunofluorescence of embryonic tissues represents a powerful application, allowing visualization of the three-dimensional organization of developing lymphatic vessels. In this context, biotin-conjugated PROX1 antibodies can be used to identify lymphatic endothelial cell progenitors budding from cardinal veins, with the biotin-streptavidin detection system providing signal amplification necessary for early developmental stages when PROX1 expression is initially low . For lineage tracing experiments, combining PROX1 immunostaining with genetic reporter systems (such as those in Prox1+/Cre models) enables researchers to track the fate of PROX1-expressing cells throughout development and confirm the specificity of the reporter system . Time-course studies throughout embryonic development (E10.5-E14.5) can reveal critical windows when PROX1 expression determines lymphatic cell fate commitment, with antibody detection confirming protein expression at these key timepoints . Co-localization studies pairing biotin-conjugated PROX1 antibodies with markers of blood vascular endothelial cells can demonstrate the mutual exclusivity of these lineages and confirm PROX1's role in lymphatic specification . For mechanistic studies investigating PROX1's interaction with Wnt signaling, immunoprecipitation using biotin-conjugated antibodies followed by Western blotting for Wnt pathway components can reveal stage-specific protein interactions driving lymphatic development .

What experimental design considerations are important when using PROX1 antibodies in cancer research?

Cancer research applications of PROX1 antibodies require carefully designed experimental approaches that account for tissue-specific expression patterns and potential pathological alterations. When examining PROX1 expression in cancer tissues, researchers should implement tissue microarray (TMA) approaches with biotin-conjugated PROX1 antibodies to efficiently screen multiple tumor samples while minimizing technical variability. Appropriate antigen retrieval methods are critical—for human lung cancer tissue, TE buffer at pH 9.0 has demonstrated optimal results, though citrate buffer at pH 6.0 provides an acceptable alternative . Expression quantification should employ digital pathology approaches with standardized scoring systems, differentiating between nuclear (transcriptionally active) and cytoplasmic PROX1 localization, which may have different prognostic implications. Cell line models should be carefully selected based on documented PROX1 expression—HuH-7 cells represent a validated positive control for PROX1 detection . For functional studies, PROX1 knockdown/overexpression paired with migration, invasion, and angiogenesis assays can reveal its role in tumor progression. Co-expression analysis pairing PROX1 with established cancer biomarkers can identify potential prognostic signatures, while survival analysis correlating PROX1 expression levels with patient outcomes can establish clinical relevance. For mechanistic investigations, ChIP-seq using biotin-conjugated PROX1 antibodies can identify cancer-specific transcriptional targets, potentially revealing therapeutic vulnerabilities in PROX1-expressing tumors.

How should researchers approach PROX1 protein-protein interaction studies using biotin-conjugated antibodies?

Protein-protein interaction studies involving PROX1 require specialized approaches that leverage the advantages of biotin conjugation while addressing potential limitations. Co-immunoprecipitation (Co-IP) experiments represent the most straightforward application—researchers can use biotin-conjugated PROX1 antibodies coupled with streptavidin-coated magnetic beads to efficiently isolate PROX1 and its interacting partners from cell or tissue lysates. This approach is particularly valuable for investigating PROX1's documented interactions with β-catenin and TCF7L1 in the context of Wnt signaling regulation . When designing Co-IP protocols, pre-clearing lysates with unconjugated streptavidin beads is essential to reduce non-specific binding. For detecting transient or weak interactions, researchers should consider implementing crosslinking steps before cell lysis using membrane-permeable crosslinkers like DSP (dithiobis(succinimidyl propionate)). Proximity ligation assays (PLA) offer an alternative approach for visualizing protein interactions in situ—combining biotin-conjugated PROX1 antibodies with antibodies against potential interaction partners, each connected to complementary DNA oligonucleotides that generate amplifiable signals when in close proximity. For more comprehensive interaction mapping, BioID or APEX2 proximity labeling can be implemented by fusing these enzymes to PROX1, followed by biotin-conjugated antibody verification of expression and localization. When analyzing interaction data, researchers should validate findings using reciprocal Co-IP experiments and include appropriate negative controls (IgG isotype and known non-interacting proteins) to confirm specificity.

What are the considerations for using PROX1 antibodies in comparative studies across different species?

Comparative studies using PROX1 antibodies across species require careful consideration of epitope conservation and validation strategies to ensure reliable cross-species reactivity. Sequence alignment analysis should be performed before selecting antibodies, focusing on the immunogen region—the biotin-conjugated polyclonal antibody targeting amino acids 2-259 of human PROX1 may have broader species reactivity due to its recognition of multiple epitopes within this conserved region . While many PROX1 antibodies have validated reactivity with human and rat samples , researchers working with other species should perform preliminary validation experiments using positive control tissues from target species. Western blot validation represents the most definitive approach, confirming both antibody reactivity and the molecular weight of detected proteins (expected at approximately 83-90 kDa) . When designing comparative immunohistochemistry experiments, species-specific optimization of antigen retrieval methods is essential—conditions optimized for human tissues may require adjustment for other species. Blocking protocols should be adapted using serum from the host species of the detection reagent rather than the antibody host species to minimize background. For evolutionary studies comparing PROX1 expression patterns across distant species, researchers should consider including both biotin-conjugated and unconjugated antibodies targeting different epitopes to confirm staining specificity. In all comparative applications, researchers should include appropriate positive and negative control tissues from each species to facilitate accurate interpretation of results and account for potential differences in PROX1 expression levels and patterns across evolutionary lineages.

How might single-cell techniques be integrated with PROX1 antibody-based detection for developmental biology research?

Single-cell approaches combined with PROX1 antibody detection represent powerful emerging tools for dissecting developmental processes with unprecedented resolution. Single-cell RNA sequencing (scRNA-seq) paired with protein verification using biotin-conjugated PROX1 antibodies can identify transcriptional states associated with lymphatic specification while confirming protein expression at the cellular level. For spatial context preservation, techniques such as Seq-Scope or Slide-seq can map PROX1 mRNA expression patterns across developing tissues, with sequential immunofluorescence using biotin-conjugated antibodies validating protein localization in the same tissue sections. Index sorting approaches allow researchers to isolate individual cells based on PROX1 immunostaining for subsequent single-cell genomic analysis, enabling direct correlation between protein expression levels and transcriptional profiles. Emerging CITE-seq (Cellular Indexing of Transcriptomes and Epitopes by Sequencing) protocols can be adapted to include biotin-conjugated PROX1 antibodies, simultaneously measuring surface protein markers, PROX1 expression, and transcriptomes in thousands of single cells. For developmental lineage tracing, integrating biotin-conjugated PROX1 antibody staining with genetic barcoding approaches can reveal the relationship between PROX1 expression timing and cell fate determination with single-cell resolution. Microfluidic systems that capture individual cells for imaging and subsequent molecular analysis represent another frontier, allowing dynamic measurement of PROX1 expression in living cells followed by downstream genomic or proteomic characterization of the same cells.

What emerging technologies might enhance the utility of biotin-conjugated PROX1 antibodies in research?

Emerging technologies promise to significantly expand the research applications of biotin-conjugated PROX1 antibodies across multiple fields. Mass cytometry (CyTOF) adapted for intracellular transcription factor detection offers the potential to simultaneously measure PROX1 expression alongside dozens of other proteins at single-cell resolution, with biotin-metal conjugates providing detection specificity. Super-resolution microscopy techniques like STORM and PALM can utilize the strong biotin-streptavidin interaction for precise localization of PROX1 within nuclear subcompartments, potentially revealing previously undetectable spatial organization of PROX1-dependent transcriptional complexes. Expanding multiplexed immunofluorescence capacity through cyclic immunofluorescence methods allows integration of biotin-conjugated PROX1 antibodies into panels with 40+ markers on single tissue sections, enabling comprehensive phenotyping of PROX1-expressing cells and their microenvironments. For in vivo applications, biotin-conjugated PROX1 antibodies could be paired with activatable fluorophores for intravital microscopy of lymphatic development in transparent organisms like zebrafish embryos. CRISPR-based technologies for genomic labeling (CROP-seq, Perturb-seq) can be validated using biotin-conjugated PROX1 antibodies to confirm knockout efficiency while simultaneously measuring transcriptional consequences. Single-molecule approaches like smFISH (fluorescence in situ hybridization) for RNA detection paired with PROX1 protein detection using biotin-conjugated antibodies can reveal the relationship between transcription and protein accumulation at subcellular resolution. Finally, adaptations for high-throughput screening applications could enable drug discovery targeted at PROX1-dependent developmental processes or pathological conditions.