HOXA2 Antibody

What is HOXA2 and why is it significant in developmental biology research?

HOXA2 functions as a sequence-specific transcription factor within a developmental regulatory system that provides cells with specific positional identities along the anterior-posterior axis. It contains a homeobox DNA-binding domain and plays a crucial role in embryonic development. In developmental pathways, HOXA2 works collaboratively with other Hox proteins such as HOXA1 and HOXA3 to modulate gene expression and help delineate segmental identities along the body axis .

The protein has been associated with microtia, hearing impairment, and cleft palate, making it an important research target for understanding craniofacial development and associated disorders . As a 376 amino acid protein belonging to the Antp homeobox family, HOXA2 has a predicted molecular weight of 41 kDa but is typically observed at 41-43 kDa in Western blot applications .

What types of HOXA2 antibodies are commercially available and what applications are they validated for?

Current commercially available HOXA2 antibodies include:

It's important to note that while these antibodies have been validated for specific applications, optimization may be required for your particular experimental system .

What are the recommended dilutions for HOXA2 antibodies in different applications?

Based on validation data, the following dilutions are recommended:

These dilutions should be considered starting points, and optimization for specific experimental conditions is advised .

How should I design proper controls when using HOXA2 antibodies in my research?

For robust experimental design with HOXA2 antibodies, incorporate these controls:

• Negative Controls: Include an isotype control antibody (e.g., Rabbit IgG) processed identically to your HOXA2 antibody samples to assess non-specific binding.

• Positive Controls: Use tissues or cell lines known to express HOXA2, such as Jurkat cells (human T cell leukemia), HepG2 cells, or C6 cells, which have been validated in previous studies .

• Molecular Weight Validation: Verify that your observed band is at the expected molecular weight (41-43 kDa) for HOXA2 protein .

• Cross-Validation: When possible, use multiple antibodies targeting different epitopes of HOXA2 to confirm specificity of detection.

• Knockdown/Knockout Validation: The gold standard for antibody validation is to include samples where HOXA2 has been knocked down or knocked out to confirm absence of signal.

What cell types and tissue samples are most suitable for HOXA2 expression studies?

Based on validation data from antibody manufacturers, these cell types and tissues have demonstrated detectable HOXA2 expression:

Cell Lines:

• Jurkat (human T cell leukemia cell line)

• HepG2 (human liver cancer cell line)

• C6 (rat glial cell line)

• U-2 OS (human bone osteosarcoma epithelial cell line)

Tissue Samples:

• Human liver cancer tissue

• Human tonsil tissue

• Mouse liver tissue

• Mouse kidney tissue

• Mouse stomach tissue

When selecting experimental models, consider that HOXA2 expression may vary based on developmental stage and tissue context .

How can I optimize Western blot protocols for detecting HOXA2?

To optimize Western blot detection of HOXA2:

• Sample Preparation: For cell lysates, use RIPA buffer with protease inhibitors. For tissues, homogenize thoroughly in cold lysis buffer to ensure complete protein extraction.

• Loading Control: Use 20-50 μg of total protein per lane, confirmed by BCA or Bradford assay. Include loading controls such as GAPDH, β-actin, or total protein stains.

• Gel Percentage: Use 10-12% SDS-PAGE gels for optimal resolution of the 41-43 kDa HOXA2 protein.

• Transfer Conditions: Transfer at 100V for 60-90 minutes using PVDF membrane (preferred over nitrocellulose for this molecular weight range).

• Blocking: Block with 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature.

• Antibody Incubation:

  • For ab229960: Dilute 1:2000 and incubate overnight at 4°C

  • For 25044-1-AP: Dilute 1:500-1:1000 and incubate overnight at 4°C

• Secondary Antibody: Use goat anti-rabbit IgG-HRP at 1:5000-1:50000 dilution .

Can HOXA2 antibodies be used in CUT&RUN assays, and what considerations are important?

While the search results don't specifically mention HOXA2 antibodies in CUT&RUN assays, this technique is applicable for transcription factors like HOXA2. For successful implementation:

• Cell Number Optimization: Start with 100,000 cells per assay, which has been shown sufficient for transcription factors. For limited samples, as few as 10,000-20,000 cells may work for transcription factors .

• Antibody Selection: Choose antibodies validated for chromatin immunoprecipitation (ChIP) as a starting point, as these often work well in CUT&RUN.

• Controls: Include both positive controls (like H3K4me3 antibody) and negative controls (IgG isotype control) to validate your experimental system .

• Target Accessibility: HOXA2 as a transcription factor may be associated with both euchromatin and heterochromatin. CUT&RUN shows no bias toward either chromatin state, making it suitable for studying HOXA2 binding sites across the genome .

• Data Analysis: For HOXA2, which regulates HOX gene clusters, analyze enrichment at known target regions like HOXA and HOXD gene clusters, similar to the approach used for other developmental transcription factors .

How does HOXA2 interact with other HOX proteins in developmental pathways?

HOXA2 functions in concert with other Hox proteins, particularly HOXA1 and HOXA3, to modulate gene expression and establish segmental identities along the body axis . This coordination creates a regulatory network that:

• Establishes Spatial Identity: The combined expression patterns of HOX proteins define positional information during embryonic development.

• Sequential Activation: HOX genes are typically activated in a sequential manner, with HOXA1, HOXA2, and HOXA3 forming part of this ordered expression pattern.

• Functional Redundancy and Specificity: While there is some functional overlap between HOX proteins, HOXA2 has specific roles that cannot be compensated by other family members.

• Target Gene Regulation: HOXA2 binds to specific DNA sequences through its homeobox domain to activate or repress target genes involved in developmental processes.

Advanced research questions might explore how modifications to HOXA2 affect its interactions with other HOX proteins or how these interactions are altered in developmental disorders .

What are the challenges in detecting HOXA2 in different experimental contexts?

Researchers face several challenges when working with HOXA2 antibodies:

• Temporal Expression Patterns: HOXA2 expression is developmentally regulated, making timing critical when studying embryonic or developmental processes.

• Spatial Heterogeneity: Expression can vary significantly between tissues and even within tissue regions, requiring careful sample selection and preparation.

• Cross-Reactivity Concerns: Due to the high homology between HOX family members, antibody cross-reactivity must be carefully evaluated.

• Post-Translational Modifications: These may affect epitope recognition, potentially leading to inconsistent detection depending on the cellular context.

• Fixation Effects: For IHC applications, the fixation method can significantly impact HOXA2 epitope accessibility. Paraffin embedding has been successfully used with ab229960 at 1:100 dilution, but optimization of antigen retrieval methods may be necessary .

Why might I observe unexpected bands in Western blot when using HOXA2 antibodies?

When unexpected bands appear in Western blots with HOXA2 antibodies, consider these potential causes and solutions:

• Protein Degradation: Fresh samples with added protease inhibitors are essential. Multiple bands below 41 kDa may indicate degradation products.

• Post-Translational Modifications: Bands slightly higher than 41 kDa (as seen with the observed 42-43 kDa bands) may represent phosphorylated, glycosylated, or otherwise modified HOXA2 protein .

• Splice Variants: Alternative splicing may generate HOXA2 isoforms of different molecular weights.

• Non-specific Binding: Increase blocking time/concentration or optimize antibody dilution to reduce non-specific signals.

• Cross-Reactivity: Due to sequence homology between HOX family members, cross-reactivity with HOXA1, HOXA3, or other HOX proteins may occur. Validate specificity using knockout/knockdown approaches.

How can I optimize immunofluorescence detection of HOXA2 in cell culture systems?

For optimal immunofluorescence detection of HOXA2:

• Fixation Protocol: PFA fixation (4%) for 15 minutes at room temperature has been validated for HOXA2 detection in U-2 OS cells .

• Permeabilization: Triton X-100 permeabilization is recommended for nuclear transcription factors like HOXA2 .

• Antibody Selection: ab222304 has been validated for ICC/IF applications at a concentration of 4 μg/ml .

• Background Reduction:

  • Increase blocking time (1-2 hours with 5% normal serum)

  • Include 0.1-0.3% Triton X-100 in blocking buffer

  • Use conjugated secondary antibodies with minimal cross-reactivity

• Signal Amplification: For low-expressing samples, consider using tyramide signal amplification or other amplification methods.

• Counterstaining: Include nuclear counterstains like DAPI to confirm nuclear localization expected for HOXA2.

• Confocal Microscopy: Use confocal microscopy for precise subcellular localization of HOXA2 within the nucleus.

How can I validate that my HOXA2 antibody is detecting the intended target?

Rigorous validation of HOXA2 antibody specificity requires multiple approaches:

• Multiple Antibodies: Use antibodies targeting different epitopes of HOXA2 (e.g., ab229960 targeting aa 1-150 and ab222304 targeting aa 200-300) and compare detection patterns .

• Genetic Approaches:

  • siRNA/shRNA knockdown of HOXA2 should decrease signal intensity

  • CRISPR/Cas9 knockout should eliminate specific signal completely

  • Overexpression systems should show increased signal intensity

• Peptide Blocking: Pre-incubate antibody with immunizing peptide to block specific binding.

• Cross-species Validation: Confirm detection in multiple species where HOXA2 is conserved (e.g., human, mouse, rat) .

• Mass Spectrometry: For definitive validation, immunoprecipitate HOXA2 and confirm identity by mass spectrometry.

What are the considerations for studying HOXA2 in the context of developmental disorders?

When investigating HOXA2 in developmental disorders:

• Tissue Selection: Focus on tissues relevant to HOXA2-associated conditions such as craniofacial structures for cleft palate studies, auditory structures for hearing impairment research, and ear development for microtia investigations .

• Developmental Timing: HOXA2 expression is temporally regulated during embryonic development. Sample collection timing is critical when using model organisms.

• Genetic Background Effects: Consider how genetic background may influence HOXA2 expression and function, particularly in mouse models.

• Human Samples: For studies using human tissues:

  • Paraffin-embedded samples have been successfully used with HOXA2 antibodies (ab229960 at 1:100 dilution)

  • Compare affected tissues with matched normal controls

  • Consider developmental stage matching between samples

• Functional Assays: Combine expression studies with functional assays to understand how HOXA2 variants affect downstream target genes and developmental processes.

How can advanced genomic techniques be combined with HOXA2 antibody applications?

Integration of genomic techniques with HOXA2 antibody applications enables comprehensive understanding of HOXA2 function:

• CUT&RUN Sequencing: While not specifically validated for HOXA2, this technique offers advantages over ChIP-seq for identifying genomic binding sites, requiring fewer cells (10,000-20,000) and providing higher signal-to-noise ratio .

• ChIP-seq: Map genome-wide binding sites of HOXA2 to identify target genes and regulatory elements.

• RNA-seq with HOXA2 Manipulation: Combine HOXA2 overexpression or knockdown with transcriptome analysis to identify genes regulated by HOXA2.

• HiChIP/PLAC-seq: Investigate 3D chromatin interactions mediated by HOXA2 to understand long-range gene regulation.

• CUT&Tag: An alternative to CUT&RUN that may offer advantages for certain experimental contexts.

• Single-cell Approaches: Combine HOXA2 antibodies with single-cell techniques to understand cell-type specific functions and heterogeneity.

When implementing these approaches, always include appropriate controls as described in the CUT&RUN protocol, including both target-specific antibody controls and negative controls .