hoxa13a Antibody

What is the HOXA13 protein and why is it important in research?

HOXA13 is a sequence-specific transcription factor belonging to the homeobox gene family that plays critical roles in embryonic development, particularly in limb (hands and feet), urinary tract, and reproductive system formation. It contains three polyalanine tracts of unknown function and primarily acts as a DNA-binding protein regulating gene expression . HOXA13 has gained significant research interest due to its involvement in developmental disorders like hand-foot-genital syndrome and various cancers, including esophageal, prostate, and nasopharyngeal carcinomas .

What types of HOXA13 antibodies are available for research, and how should I select one?

Researchers can choose from several types of HOXA13 antibodies:

Selection criteria should include: (1) validated applications matching your experimental needs, (2) species reactivity relevant to your model system, (3) epitope location that doesn't interfere with protein interactions of interest, and (4) conjugation status (unconjugated vs. labeled with HRP, fluorophores, etc.) .

How do HOXA13 and HOXB13 antibodies differ, and can they cross-react?

While HOXA13 and HOXB13 are paralogs within the HOX gene family with some functional similarities, they represent distinct proteins with different expression patterns. HOXA13 shows higher expression in Barrett's esophagus compared to HOXB13 . Cross-reactivity is a significant concern due to the high homology within the homeodomain region. When selecting an antibody, researchers should:

• Examine the immunogen sequence to ensure it targets unique regions

• Review validation data showing specificity testing

• Consider performing knockdown/knockout controls in their specific experimental system

• Verify antibody specificity by comparing staining patterns with known expression profiles

What are the optimal conditions for using HOXA13 antibodies in Western blotting?

For optimal Western blot results with HOXA13 antibodies:

• Sample preparation:

  • Nuclear extracts are often preferable as HOXA13 is primarily nuclear

  • LNCaP and PC-3 cells serve as positive controls for human samples

• Running conditions:

  • 12% SDS-PAGE gels are suitable for resolving HOXA13 (30-39 kDa)

  • Include molecular weight markers spanning 25-50 kDa

• Transfer and detection:

  • PVDF membranes typically yield better results than nitrocellulose

  • Blocking with 5% non-fat milk or BSA in TBST (1 hour at room temperature)

  • Primary antibody dilutions ranging from 1:500-1:1000 are typically effective

  • Incubate with HRP-conjugated secondary antibody and develop using ECL

Expected band size is 30-39 kDa, with some variation depending on post-translational modifications and the cell/tissue type being analyzed .

How can I optimize HOXA13 antibody-based immunohistochemistry protocols?

For successful immunohistochemical detection of HOXA13:

• Tissue preparation:

  • Formalin-fixed paraffin-embedded sections (5 μm thickness)

  • Fresh-frozen sections may provide higher sensitivity but poorer morphology

• Antigen retrieval:

  • Heat-induced epitope retrieval using TE buffer pH 9.0 yields optimal results

  • Alternative: citrate buffer pH 6.0 with slightly reduced sensitivity

• Staining protocol:

  • Use antibody concentrations of 1-10 μg/mL or dilutions of 1:50-1:500

  • Incubate overnight at 4°C for maximal sensitivity

  • DAB detection systems work effectively for HOXA13 visualization

  • Counterstain with hematoxylin for nuclear contrast

• Controls:

  • Include known positive tissues (embryonic limb buds, prostate tissue)

  • Use sections from HOXA13 knockout models as negative controls where available

Note that some researchers report difficulties with HOXA13 immunohistochemistry, requiring in situ hybridization as an alternative approach .

What techniques beyond standard immunoassays can be used to study HOXA13 protein interactions and function?

Advanced techniques for studying HOXA13 include:

• Chromatin Immunoprecipitation (ChIP):

  • Successfully performed with HOXA13 antibodies in embryonic tissues

  • Requires cross-linking with 1% formaldehyde (10 min at 4°C)

  • Sonication to generate 200-1000 bp fragments

  • 11-18 pairs of embryonic tissues provide sufficient material

• Co-Immunoprecipitation (Co-IP):

  • Effective for studying HOXA13 protein-protein interactions

  • Nuclear extracts provide higher yields than whole cell lysates

  • Gentle lysis conditions preserve protein complexes

• ChIP-seq:

  • Reveals genome-wide binding profiles of HOXA13

  • Protocol modifications include extended cross-linking times (15 min)

  • Requires high-quality antibodies validated for ChIP applications

• RNA-interference combined with antibody detection:

  • siRNA knockdown of HOXA13 followed by antibody-based detection

  • Provides functional validation of antibody specificity

  • Reveals downstream effects of HOXA13 depletion

What are common problems with HOXA13 antibodies and how can they be addressed?

Common challenges with HOXA13 antibodies include:

• Lack of specificity:

  • Solution: Test multiple antibodies targeting different epitopes

  • Validate using positive and negative controls (tissues with known expression)

  • Confirm with RNA interference or gene editing approaches

• High background in immunostaining:

  • Solution: Optimize blocking (try 2-5% BSA or 10% normal serum)

  • Increase washing steps (5x 5 min washes)

  • Decrease primary antibody concentration

  • Use antibody diluent containing 0.1-0.3% Triton X-100

• Failed immunohistochemistry:

  • Some researchers report difficulties with HOXA13 IHC

  • Alternative: Use in situ hybridization for HOXA13 mRNA detection

  • Try different antigen retrieval methods (heat vs. enzymatic)

• Nuclear versus cytoplasmic localization discrepancies:

  • HOXA13 can exhibit both nuclear (primary) and cytoplasmic localization

  • Verify fixation conditions don't cause artifactual redistribution

  • Include cellular fractionation controls in Western blot analyses

How can I validate the specificity of a HOXA13 antibody for my research?

A comprehensive validation approach includes:

• Positive and negative controls:

  • Use tissues/cells with confirmed HOXA13 expression (prostate, embryonic limb buds)

  • Include HOXA13-knockout or knockdown samples

  • Compare with in situ hybridization patterns when possible

• Peptide competition assays:

  • Pre-incubate antibody with immunizing peptide

  • Should abolish specific signal while non-specific binding remains

• Orthogonal methods:

  • Compare protein detection with mRNA expression (qPCR, RNA-seq)

  • Verify subcellular localization matches known distribution pattern

• Cross-reactivity testing:

  • Test antibody against recombinant HOXA13 and related HOX proteins

  • Particularly important to distinguish from HOXB13

  • Western blot for predicted molecular weight (30-39 kDa)

What controls should be included when using HOXA13 antibodies in different experimental setups?

Essential controls for HOXA13 antibody experiments include:

Additionally, for developmental studies, stage-matched wild-type and mutant tissues provide critical comparative controls .

How can HOXA13 antibodies be used to investigate cancer progression mechanisms?

HOXA13 antibodies have revealed significant insights into cancer biology:

• Expression analysis in tumor progression:

  • HOXA13 overexpression correlates with poor survival in esophageal cancer

  • IHC with HOXA13 antibodies can stratify patients into prognostic groups

  • Sequential samples can track HOXA13 expression changes during tumor evolution

• Molecular mechanism studies:

  • ChIP-seq with HOXA13 antibodies identifies cancer-relevant target genes

  • Co-IP reveals interactions with cancer signaling proteins

  • Combined with RNA-seq after HOXA13 knockdown to identify regulatory networks

• EMT and invasion pathways:

  • HOXA13 promotes epithelial-to-mesenchymal transition in multiple cancers

  • Antibodies help track changes in HOXA13 nuclear localization during EMT

  • Co-staining with EMT markers (Snail, MMP-2) reveals mechanistic links

  • HOXA13 has been shown to affect Wnt and TGF-β pathways in glioma

• Therapeutic target validation:

  • Antibodies validate HOXA13 knockdown efficiency in preclinical models

  • Help correlate HOXA13 levels with treatment response

What insights have been gained about HOXA13 function through antibody-based approaches in developmental biology?

Antibody-based studies have revealed critical aspects of HOXA13 developmental function:

• Spatial-temporal expression patterns:

  • HOXA13 shows precise expression boundaries in developing embryos

  • Antibody staining revealed HOXA13 expression at the gastroesophageal junction

  • Single HOXA13-positive cells identified distal from physiological esophagus

• Regulatory mechanisms:

  • ChIP studies identified HOXA13 binding to regulatory regions of BMP2 and BMP7

  • HOXA13 regulates autopod-specific expression of Hoxd13

  • Interactions with TGF-β/Activin-regulated Smad proteins detected

• Cellular distribution patterns:

  • Nuclear localization predominates in most cell types

  • Cytoplasmic localization observed during fetal skin development

  • Gradient distribution along the proximal-distal and baso-luminal axes of crypts

• Disease mechanisms:

  • Mutations affecting polyalanine tracts cause protein degradation

  • Single amino acid substitutions may affect protein folding and DNA binding

  • These insights help explain hand-foot-genital syndrome pathogenesis

How can HOXA13 antibodies be incorporated into single-cell analysis techniques?

Integrating HOXA13 antibodies into single-cell techniques enables:

• Single-cell protein analysis:

  • Flow cytometry with HOXA13 antibodies identifies subpopulations

  • Mass cytometry (CyTOF) allows multiplexed detection of HOXA13 with other markers

  • Imaging mass cytometry reveals spatial context of HOXA13-expressing cells

• Spatial transcriptomics correlation:

  • Combining in situ hybridization for HOXA13 mRNA with antibody detection

  • Co-registration with single-cell RNA-seq data

  • Revealed HOXA13-positive cells in normal squamous esophagus (8%) and Barrett's esophagus (30%)

• Lineage tracing applications:

  • Antibodies help validate HOXA13 reporter systems

  • HOXA13-GFP reporter mice show clonal expression patterns at crypt borders

  • Single HOXA13-positive cells identified at gastroesophageal junction

• Cell fate determination studies:

  • HOXA13-expressing cells show competitive advantage

  • Expression increases in certain pathologies without changes in per-cell expression level

  • Suggests expansion of HOXA13-positive compartment rather than upregulation

What is known about HOXA13 protein interactions with other transcription factors and signaling pathways?

HOXA13 engages in multiple protein interactions affecting various signaling pathways:

• Interactions with other transcription factors:

  • HOXA13 interacts with the DNA binding domain of androgen receptor (AR)

  • Functions as both a positive and negative regulator of AR target genes

  • Cluster 2 genes (including PSA/KLK3, KLK2, KLK4, FASN) are repressed by HOXA13

  • Cluster 3 genes (TMPRSS2, NKX3.1, PMEPA1) require HOXA13 for androgen responsiveness

• Signaling pathway involvement:

  • Wnt signaling: HOXA13 decreases β-catenin in the nucleus and increases phospho-β-catenin in cytoplasm

  • TGF-β pathway: HOXA13 decreases phospho-SMAD2 and phospho-SMAD3 in the nucleus

  • BMP signaling: HOXA13 and HOXD13 bind to BMP/TGF-β-regulated Smad proteins (Smad1, Smad2)

  • EMT regulation: HOXA13 upregulates Snail and MMP-2 expression in nasopharyngeal carcinoma

• DNA binding characteristics:

  • HOXA13 has AT-rich binding preferences

  • Can bind through direct DNA interaction or through association with other transcription factors

  • HOXB13 binds preferentially to methylated DNA

  • DNA binding-deficient HOXA13 (HOXA13-3A) retains repressor function on some genes

How do HOXA13 expression patterns differ across tissues and developmental stages?

HOXA13 shows distinct spatial and temporal expression patterns:

• Developmental expression:

  • Critical for limb (particularly hands/feet) development

  • Essential for urinary tract and reproductive system formation

  • Present in human embryonic esophagus during columnar-to-squamous transition (17-20 weeks gestation)

  • High expression at gastric cardia in human fetuses

• Adult tissue distribution:

  • Tightly regulated along gastrointestinal tract

  • Adult squamous esophagus shows low expression

  • Single HOXA13-positive cells present at gastroesophageal junction

  • Expression increases from ileocecal valve to distal transverse colon

  • Present in esophageal submucosal glands (ESMG)

• Subcellular localization patterns:

  • Primarily nuclear in most tissues

  • Cytoplasmic expression during fetal skin development

  • In colonic crypts, distribution varies along baso-luminal axis (apical expression proximally, entire crypt distally)

• Pathological expression:

  • Increased in Barrett's esophagus and esophageal adenocarcinoma

  • Overexpressed in nasopharyngeal carcinoma, promoting proliferation, migration, and invasion

  • Expanded population of HOXA13-positive cells rather than increased per-cell expression

What are the implications of HOXA13 research for understanding human disease pathogenesis?

HOXA13 research has significant implications for understanding disease mechanisms:

• Developmental disorders:

  • Hand-foot-genital syndrome results from HOXA13 mutations

  • Polyalanine tract expansions lead to protein instability and degradation

  • Amino acid substitutions alter protein folding and function

  • These findings provide molecular basis for clinical manifestations

• Cancer biology:

  • HOXA13 is a negative independent predictor of disease-free survival in esophageal cancer

  • Promotes cell growth, inhibits apoptosis, and enhances invasion

  • Knockdown of HOXA13 significantly reduces tumor growth in vivo

  • Involved in Barrett's esophagus pathogenesis, a precursor to esophageal adenocarcinoma

  • Promotes nasopharyngeal carcinoma through Snail and MMP-2 upregulation

  • Functions as an oncogene in glioma by activating Wnt/TGF-β pathways

• Tissue metaplasia:

  • HOXA13 expression in Barrett's esophagus explains both phenotype (through downregulation of epidermal differentiation complex) and oncogenic potential

  • Single HOXA13-positive cells may represent origin cells for metaplasia

  • HOXA13 expression confers competitive advantage to cells

• Therapeutic implications:

  • Potential diagnostic biomarker for glioblastoma

  • Independent prognostic factor in high-grade glioma

  • Targeting HOXA13 or its downstream pathways represents a potential therapeutic strategy

How do different fixation and sample preparation methods affect HOXA13 antibody performance?

Sample preparation significantly impacts HOXA13 antibody performance:

• Fixation considerations:

  • Formaldehyde (1%, 10 min at 4°C) works effectively for ChIP applications

  • For immunofluorescence, 4% paraformaldehyde (10-15 min) preserves epitope accessibility

  • Methanol fixation (80%, 5 min) followed by permeabilization works for flow cytometry

  • Overfixation may mask epitopes and reduce antibody binding

• Tissue processing:

  • Fresh frozen sections typically provide higher sensitivity but poorer morphology

  • FFPE sections require optimization of antigen retrieval (TE buffer pH 9.0 recommended)

  • Nuclear HOXA13 detection requires adequate permeabilization (0.1-0.3% Triton X-100)

• Cell preparation:

  • For nuclear proteins like HOXA13, nuclear extraction protocols yield cleaner results

  • Dounce homogenization (13 strokes with tight-fitting homogenizer) works well for embryonic tissues

  • Cell lysis conditions must balance efficient extraction with preservation of protein-protein interactions

What emerging technologies might enhance HOXA13 protein research in the near future?

Emerging technologies with potential to advance HOXA13 research include:

• Advanced imaging approaches:

  • Super-resolution microscopy to visualize HOXA13 nuclear distribution patterns

  • Live-cell imaging with tagged HOXA13 to track dynamic protein movements

  • Multiplexed ion beam imaging (MIBI) for simultaneous detection of numerous proteins

• Integrative multi-omics:

  • Combined ChIP-seq and ATAC-seq to correlate HOXA13 binding with chromatin accessibility

  • Integration of proteomics and transcriptomics to identify complete HOXA13 regulatory networks

  • Single-cell multi-omics to profile HOXA13 at transcriptomic and proteomic levels simultaneously

• Protein engineering approaches:

  • Nanobodies against HOXA13 for improved imaging and in vivo applications

  • CRISPR-based tagging of endogenous HOXA13 to avoid overexpression artifacts

  • Proximity labeling techniques (BioID, APEX) to map complete HOXA13 interactomes

• Therapeutic development:

  • Antibody-drug conjugates targeting HOXA13-expressing cancer cells

  • Small molecule inhibitors of HOXA13-protein interactions

  • RNA therapeutics to modulate HOXA13 expression in disease contexts

What interdisciplinary approaches might benefit from HOXA13 antibody applications?

Cross-disciplinary applications of HOXA13 antibodies include:

• Developmental biology and regenerative medicine:

  • Tracking HOXA13 expression during tissue regeneration

  • Monitoring differentiation status of stem cells in culture

  • Validating tissue engineering approaches for limb and urogenital reconstruction

• Cancer biology and precision medicine:

  • HOXA13 as a biomarker for patient stratification

  • Monitoring treatment response through HOXA13 expression changes

  • Companion diagnostics for therapies targeting HOXA13-dependent pathways

• Evolutionary developmental biology:

  • Comparative analysis of HOXA13 expression across species

  • Understanding conservation and divergence of HOX gene functions

  • Correlating morphological innovations with HOXA13 expression patterns

• Systems biology:

  • Network modeling of HOXA13 transcriptional regulation

  • Integration of protein interactome data with transcriptional outputs

  • Computational prediction of HOXA13 binding sites validated by ChIP-seq