nkx2.5 Antibody, Biotin conjugated

What is NKX2.5 and why is it significant in cardiovascular research?

NKX2.5 is a homeodomain-containing transcription factor that plays critical roles in regulating tissue-specific gene expression essential for tissue differentiation and determining temporal and spatial patterns of development. This transcription factor is particularly implicated in congenital heart disease and is required for regulation of second heart field (SHF) progenitors contributing to cardiac development . NKX2.5 has a calculated molecular weight of 35 kDa based on its 324 amino acid sequence, though it is typically observed at a range of 30-42 kDa in experimental conditions . The gene encoding NKX2.5 (also known as NK2 transcription factor related, locus 5) is highly conserved across species, indicating its fundamental importance in cardiac morphogenesis. Researchers investigate NKX2.5 to understand the molecular mechanisms underlying heart development and the pathogenesis of congenital heart defects.

What are the primary research applications for biotinylated NKX2.5 antibodies?

Biotinylated NKX2.5 antibodies serve multiple research applications with particular strengths in specific techniques:

• Immunohistochemistry (IHC): These antibodies excel in detecting NKX2.5 in fixed tissue sections, particularly human heart tissue, allowing visualization of expression patterns within the tissue architecture .

• Western Blot analysis: Biotinylated antibodies can be used to detect and quantify NKX2.5 protein expression in tissue or cell lysates .

• Chromatin Immunoprecipitation (ChIP): Though not explicitly mentioned for the biotinylated version, NKX2.5 antibodies have been successfully used in ChIP analyses to identify direct binding of NKX2.5 to regulatory elements in target genes .

The biotinylation allows for enhanced detection sensitivity through the strong biotin-streptavidin interaction, which is particularly valuable when studying transcription factors like NKX2.5 that may be expressed at relatively low levels.

What species reactivity can researchers expect from NKX2.5 antibodies?

The biotinylated NKX2.5 antibody (BAF2444) demonstrates specific reactivity with human NKX2.5, as it was generated using E. coli-derived recombinant human NKX2.5 (amino acids Gln24-Arg137, Accession # P52952) as the immunogen . For researchers working with other species, it's noteworthy that other NKX2.5 antibodies, such as the polyclonal antibody 13921-1-AP, show reactivity with both human and mouse samples, and have been cited for use with rat samples as well . The high degree of conservation in the NKX2.5 sequence across species means that antibodies may cross-react with orthologs from species not explicitly tested, but empirical validation is always recommended for specific experimental systems.

How should researchers optimize immunohistochemistry protocols with biotinylated NKX2.5 antibodies?

For optimal immunohistochemistry results with biotinylated NKX2.5 antibodies, researchers should consider the following protocol optimizations:

• Tissue preparation: Immersion-fixed paraffin-embedded sections have shown successful results, particularly with human heart tissue .

• Antibody concentration: A concentration of approximately 15 μg/mL has proven effective in published studies .

• Incubation conditions: Overnight incubation at 4°C appears optimal for specific binding .

• Detection system: The HRP-DAB system (such as Anti-Goat HRP-DAB Cell & Tissue Staining Kit) with hematoxylin counterstaining has been validated for visualization .

• Antigen retrieval: While specific conditions for the biotinylated version aren't detailed, other NKX2.5 antibodies recommend TE buffer pH 9.0, with citrate buffer pH 6.0 as an alternative .

• Controls: Include negative controls omitting the primary antibody to confirm staining specificity, as demonstrated in the literature where "lower panel shows a lack of labeling if primary antibodies are omitted and tissue is stained only with secondary antibody" .

Each step should be systematically optimized for specific experimental conditions and tissue sources.

What are the appropriate dilutions for different applications of NKX2.5 antibodies?

The optimal dilution of NKX2.5 antibodies varies by specific application and antibody format:

• For the biotinylated NKX2.5 antibody, specific dilution information isn't extensively detailed, but literature demonstrates successful use at 15 μg/mL for immunohistochemistry applications .

• For related NKX2.5 antibodies, the following ranges are recommended:

  • Western Blot: 1:500-1:3000 dilution

  • Immunohistochemistry: 1:50-1:500 dilution

  • Immunofluorescence: 1:50-1:500 dilution

It's emphasized in the literature that "this reagent should be titrated in each testing system to obtain optimal results" and that optimal dilutions can be "sample-dependent" . Therefore, researchers should perform dilution series experiments to determine the optimal concentration for their specific experimental conditions, tissue types, and detection systems.

How can researchers validate the specificity of NKX2.5 antibodies?

Validating antibody specificity is crucial for reliable research results. A comprehensive validation approach includes:

• Positive control tissues: Use tissues known to express NKX2.5, such as human heart tissue, which is recommended in literature as a positive control .

• Negative control tissues: Include tissues where NKX2.5 is not expressed or is expressed at very low levels.

• Knockout/knockdown validation: Some NKX2.5 antibodies have been validated in KD/KO applications according to published studies .

• Peptide competition: Pre-incubation of the antibody with its immunizing peptide (in this case, recombinant human NKX2.5 Gln24-Arg137) should eliminate specific staining.

• Secondary-only controls: For immunohistochemistry, include control staining with secondary antibody only, as demonstrated in immunohistochemistry studies where "lower panel shows a lack of labeling if primary antibodies are omitted" .

• Western blot validation: Verify that the observed molecular weight matches the expected range (30-42 kDa for NKX2.5) .

A systematic implementation of these validation strategies will ensure that experimental signals truly represent NKX2.5 rather than non-specific binding.

What controls should be included when performing immunohistochemistry with biotinylated NKX2.5 antibodies?

A comprehensive control strategy for immunohistochemistry with biotinylated NKX2.5 antibodies should include:

• Primary antibody omission control: Stain sections with detection reagents only, excluding the primary antibody, to assess background and non-specific binding of the detection system .

• Positive tissue control: Include human heart tissue, which is documented to express NKX2.5 and serves as a reliable positive control .

• Endogenous biotin blocking control: Since the antibody is biotinylated, include controls with and without endogenous biotin blocking to assess the contribution of endogenous biotin to background signal.

• Isotype control: Include sections stained with non-specific IgG of the same host species and concentration as the primary antibody to evaluate non-specific binding.

• Absorption control: Pre-incubate the antibody with recombinant NKX2.5 protein to confirm staining specificity.

• Serial dilution controls: Include sections stained with a range of antibody concentrations to determine optimal signal-to-noise ratio.

These controls collectively ensure that the observed staining pattern specifically represents NKX2.5 expression rather than technical artifacts.

How does NKX2.5 binding to promoter regions affect gene expression in cardiac development?

NKX2.5 functions as a critical transcription factor with complex regulatory roles in cardiac development:

• Dual regulatory capacity: Research has demonstrated that NKX2.5 can both activate and repress gene expression. Loss of NKX2.5 expression in null embryos led to downregulation of certain genes and upregulation of others, indicating context-dependent regulatory functions .

• Direct binding to regulatory elements: NKX2.5 binds to NK homeodomain binding elements (NKEs) with the consensus sequence TNNAGTG in both promoter and intronic regions of target genes .

• Target gene example: NKX2.5 was found to directly repress Jarid2 expression through binding to NKEs in the promoter region (-203) and the second intron (+1872) .

• Tissue-specific regulation: This binding and regulatory activity was observed to be tissue-specific, occurring in pharyngeal arch (PA) cells but not in heart cells at the E9.5 stage of development .

• Developmental stage dependency: The regulatory impact of NKX2.5 varies across developmental stages, with differential expression patterns identified between E9.5, E10.5, and E12.5 embryonic stages .

These findings highlight NKX2.5's sophisticated role in orchestrating gene expression networks essential for proper cardiac morphogenesis and function.

How can NKX2.5 antibodies be used in ChIP experiments to study direct transcriptional regulation?

Chromatin Immunoprecipitation (ChIP) with NKX2.5 antibodies provides powerful insights into direct transcriptional regulation:

• Target identification: ChIP can identify direct NKX2.5 binding to specific NK homeodomain binding elements (NKEs) with the consensus sequence TNNAGTG in both promoter and intronic regions of target genes .

• Methodology: The approach involves isolating chromatin from relevant tissues (such as E9.5 PA tissue), performing immunoprecipitation with NKX2.5 antibodies, and then conducting PCR to detect enrichment of specific DNA sequences containing potential NKX2.5 binding sites .

• Quantification: Quantitative PCR provides fold enrichment data, with studies showing a 4.5-fold enrichment of specific NKE sites in target samples .

• Essential controls: Appropriate controls include immunoprecipitation with non-immune IgG and PCR targeting regions without NKX2.5 binding sites .

• Tissue specificity analysis: ChIP experiments have revealed tissue-specific binding patterns, with NKX2.5 associating with regulatory elements in pharyngeal arch cells but not in heart cells at certain developmental stages .

For genome-wide analyses, ChIP followed by next-generation sequencing (ChIP-seq) extends this approach to identify all genomic regions bound by NKX2.5, facilitating the construction of comprehensive gene regulatory networks governing cardiac development.

What is the significance of studying NKX2.5 binding to both promoter and intronic regions?

The discovery that NKX2.5 binds to regulatory elements in both promoter and intronic regions has profound implications for understanding gene regulation:

• Multiple regulatory mechanisms: NKX2.5 can regulate gene expression through both proximal (promoter) and distal (intronic) regulatory elements, suggesting sophisticated control mechanisms .

• Tissue-specific regulatory patterns: Research has demonstrated that NKX2.5 binding to the Jarid2 gene occurs at both the promoter region (-203) and within the second intron (+1872), but this binding pattern is tissue-specific, occurring in PA cells but not heart cells at E9.5 .

• Comprehensive regulatory landscape: Understanding these complex binding patterns is essential for deciphering the complete gene regulatory networks governing cardiac development.

• Methodological implications: These findings highlight the importance of examining both promoter and non-promoter regions when studying transcription factor binding through techniques like ChIP.

• Developmental context: The differential binding patterns may contribute to the precise temporal and spatial regulation of gene expression during heart development.

This comprehensive approach to studying NKX2.5 binding provides deeper insights into its regulatory mechanisms than focusing solely on promoter regions, revealing the complex interplay between multiple regulatory elements in controlling gene expression.

How can NKX2.5 antibodies be used to study congenital heart defects?

NKX2.5 antibodies offer valuable approaches for investigating congenital heart defects:

• Expression pattern analysis: Comparative immunohistochemistry of heart tissue samples from normal and diseased states can reveal alterations in NKX2.5 expression patterns, potentially identifying dysregulation associated with congenital defects .

• Stem cell models: Analysis of NKX2.5 expression in patient-derived induced pluripotent stem cells (iPSCs) differentiated into cardiac lineages can reveal how mutations affect cardiac progenitor specification and differentiation.

• Target gene identification: ChIP approaches using NKX2.5 antibodies can identify altered downstream targets in disease models compared to normal controls, providing insights into dysregulated pathways .

• Protein level assessment: Western blot analysis of NKX2.5 protein levels in tissue from animal models of congenital heart defects can quantify expression differences.

• Functional studies: Combining NKX2.5 expression analysis with functional assays can correlate transcription factor abnormalities with specific cardiac malformations.

These approaches collectively help elucidate how NKX2.5 mutations or dysregulation contribute to the development of congenital heart defects, potentially identifying new therapeutic targets or diagnostic markers.

Why might researchers observe varying molecular weights for NKX2.5 in Western blots?

The variation in observed molecular weights for NKX2.5 in Western blots (typically 30-42 kDa range versus the calculated 35 kDa) can be attributed to several factors:

• Post-translational modifications: Phosphorylation, SUMOylation, ubiquitination, or other modifications can alter the apparent molecular weight of NKX2.5.

• Alternative splicing: Different isoforms resulting from alternative splicing of the NKX2.5 gene may exhibit different molecular weights.

• Proteolytic processing: Partial proteolytic degradation during sample preparation can generate fragments of varying sizes.

• Tissue-specific modifications: The pattern of post-translational modifications may vary between tissue types or developmental stages.

• Gel conditions: Running conditions, buffer composition, and gel percentage can affect protein migration patterns.

To address this variability, researchers should include appropriate positive controls (such as human heart tissue ) and validate band specificity using additional techniques such as immunoprecipitation or mass spectrometry. Careful standardization of sample preparation protocols can also minimize technical variability in observed molecular weights.

What are the considerations for using biotinylated NKX2.5 antibodies in tissues with high endogenous biotin?

When using biotinylated antibodies in tissues with high endogenous biotin content (such as liver, kidney, and brain), researchers should implement specific strategies to minimize background:

• Biotin blocking: Incorporate a biotin blocking step before applying the biotinylated primary antibody, using a commercially available biotin blocking kit.

• Negative controls: Include sections stained with only the detection reagents without primary antibody to assess endogenous biotin contribution to background .

• Alternative detection methods: For tissues with very high endogenous biotin, consider using non-biotinylated NKX2.5 antibodies with alternative detection systems.

• Optimized fixation: Certain fixation protocols may help reduce accessibility of endogenous biotin while preserving NKX2.5 epitopes.

• Reduced avidin/streptavidin concentration: Titrate the detection reagent concentration to find the optimal balance between specific signal and background.

• Alternative tissues: When possible, select tissue regions with lower endogenous biotin for analysis of NKX2.5 expression.

These precautions will help ensure that the observed staining truly represents NKX2.5 expression rather than endogenous biotin detection.

What are the methodological differences between using biotinylated vs. fluorophore-conjugated NKX2.5 antibodies?

The choice between biotinylated and fluorophore-conjugated NKX2.5 antibodies carries important methodological implications:

• Detection systems: Biotinylated antibodies require a secondary detection step with streptavidin/avidin conjugates (like the HRP-DAB system ), while fluorophore-conjugated antibodies (such as Alexa Fluor 647 conjugates ) provide direct detection.

• Sensitivity considerations: The biotin-streptavidin system offers signal amplification, potentially providing higher sensitivity for low-abundance transcription factors like NKX2.5, while direct fluorophore conjugation often provides cleaner background but potentially lower sensitivity.

• Application suitability: Fluorophore-conjugated antibodies are essential for flow cytometry applications and multi-color immunofluorescence, while biotinylated antibodies excel in immunohistochemistry with chromogenic detection.

• Stability differences: Fluorophores can be subject to photobleaching (requiring protection from light ), while biotinylated antibodies are generally more stable but can be affected by endogenous biotin.

• Multiplexing capability: Fluorophore-conjugated antibodies allow for more straightforward multiplexing with other antibodies in the same sample due to direct visualization.

These differences should guide the choice between biotinylated and fluorophore-conjugated NKX2.5 antibodies based on the specific research application, required sensitivity, and detection system compatibility.

What are the best storage conditions for maintaining the activity of NKX2.5 antibodies?

Optimal storage conditions are essential for maintaining antibody activity:

• Temperature: Store NKX2.5 antibodies at -20°C for long-term stability .

• Buffer composition: NKX2.5 antibodies are typically stored in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 .

• Aliquoting considerations: While aliquoting is noted as "unnecessary for -20°C storage" for some NKX2.5 antibodies, it may still be beneficial for antibodies used infrequently to minimize freeze-thaw cycles.

• Special precautions for conjugates: For fluorophore-conjugated antibodies, additional precautions include protecting from light and avoiding freezing .

• Stability timeline: When properly stored, NKX2.5 antibodies are typically stable for one year after shipment .

• Contamination prevention: Maintain sterile conditions when handling antibodies to prevent microbial growth and degradation.

Following these storage recommendations will help ensure consistent antibody performance and reproducible experimental results over time.

How can ChIP-seq with NKX2.5 antibodies be used to construct cardiac gene regulatory networks?

ChIP-seq technology with NKX2.5 antibodies offers powerful approaches for building comprehensive cardiac gene regulatory networks:

• Genome-wide binding identification: ChIP-seq expands the targeted ChIP approach demonstrated in the literature to identify all genomic regions bound by NKX2.5, including both promoter and distal regulatory elements.

• Motif analysis: Bioinformatic examination of sequences surrounding NKX2.5 binding sites can identify the consensus NK homeodomain binding elements (NKEs) with the sequence TNNAGTG and potential co-occurring transcription factor binding motifs.

• Integration with expression data: Correlating NKX2.5 binding patterns with RNA-seq data from the same tissues/developmental stages can establish direct relationships between binding events and gene expression changes.

• Developmental dynamics: Performing ChIP-seq across multiple developmental timepoints can reveal how NKX2.5-mediated regulation evolves during cardiac development.

• Tissue-specific binding: Comparing binding patterns across different cardiac regions or cell types can identify tissue-specific regulatory mechanisms, as suggested by the differential binding observed between PA cells and heart cells .

• Disease model comparison: Comparing NKX2.5 binding patterns between normal and disease models can identify dysregulated regulatory networks in congenital heart defects.

These approaches collectively enable the construction of comprehensive gene regulatory networks governing cardiac development and function, providing deeper insights into both normal development and pathological conditions.

How can single-cell analysis techniques be combined with NKX2.5 antibodies to study cardiac progenitor heterogeneity?

Integrating NKX2.5 antibody detection with single-cell technologies offers powerful approaches for dissecting cardiac progenitor heterogeneity:

• Single-cell protein and RNA profiling: Techniques like CITE-seq can simultaneously measure NKX2.5 protein expression (using conjugated antibodies similar to the Alexa Fluor 647 conjugate ) and transcriptome-wide gene expression in individual cells.

• Flow cytometry with index sorting: NKX2.5 antibody-based cell sorting combined with single-cell transcriptomics can correlate protein expression levels with global gene expression patterns at the individual cell level.

• Spatial transcriptomics: Combining immunofluorescence detection of NKX2.5 with spatial transcriptomics can map both protein expression and transcriptional profiles while preserving tissue architecture.

• Lineage tracing: Combining NKX2.5 antibody detection with genetic lineage tracing can reveal the developmental trajectories and fates of different NKX2.5-expressing progenitor subpopulations.

• Chromatin accessibility: Integrating NKX2.5 protein detection with single-cell ATAC-seq can correlate protein expression with chromatin state at the single-cell level.

These integrated approaches can reveal previously unrecognized heterogeneity within NKX2.5-expressing cardiac progenitor populations, potentially identifying new subpopulations with distinct developmental potentials or responses to disease conditions.