HOXA11/HOXD11 Antibody

Basic Research Questions

• What are the recommended applications for HOXA11/HOXD11 antibodies?

  HOXA11/HOXD11 antibodies have been validated for multiple applications with specific dilution recommendations:

  Application	Recommended Dilution	Validation Status
  Western Blot (WB)	1:500-1:2000	Validated in mouse and human samples
  Immunocytochemistry/Immunofluorescence (ICC/IF)	1:200-1:1000	Validated in HUVEC cells
  Immunohistochemistry (IHC)	1:100-1:300	Validated
  ELISA	1:20000	Validated

  For optimal results, perform application-specific validation in your experimental system. For example, in Western blot analysis, the HOXA11 antibody ab72591 shows a band at approximately 30 kDa when used at 1/500 dilution with RAW264.7 cell extracts, despite a predicted size of 34 kDa .

• What species reactivity do HOXA11/HOXD11 antibodies typically exhibit?

  Most commercially available HOXA11/HOXD11 antibodies demonstrate reactivity with human and mouse samples . This cross-reactivity stems from high sequence conservation in the homeodomain region. When planning experiments with other species, consider:

  • Performing sequence alignment analysis to predict cross-reactivity

  • Including proper positive controls from validated species

  • Running pilot experiments at multiple antibody concentrations

  Some antibodies may work with additional species based on homology, but this typically requires experimental validation and may not be covered by manufacturer guarantees .

• What are the optimal storage conditions for HOXA11/HOXD11 antibodies?

  For maximum stability and activity retention:

  • Store antibodies at -20°C in small aliquots to avoid repeated freeze/thaw cycles

  • Most formulations contain 50% glycerol with 0.5% BSA and 0.02% sodium azide as preservatives

  • Working dilutions should be prepared fresh and used within 24 hours

  • Monitor antibody performance over time using consistent positive controls

  Antibody degradation may manifest as decreased signal intensity, increased background, or appearance of non-specific bands in Western blots.

• What are the key characteristics of commonly used HOXA11/HOXD11 antibodies?

  Characteristic	HOXA11 Antibody (ab72591)	HOXA11/HOXD11 Antibody (PACO06560)
  Host	Rabbit	Rabbit
  Clonality	Polyclonal	Polyclonal
  Immunogen	Synthetic peptide within Human HOXA11	C-terminal region of human HoxA11/D11
  Isotype	IgG	IgG
  Form	Liquid	Liquid in PBS with 50% glycerol
  Purification	Not specified	Affinity chromatography

  When selecting between single-specificity (HOXA11 only) versus dual-specificity (HOXA11/HOXD11) antibodies, consider your experimental goals and the potential for cross-reactivity with other HOX proteins .

Advanced Research Questions

• How can researchers validate the specificity of HOXA11/HOXD11 antibodies in their experimental systems?

  Comprehensive validation should include:

  • Peptide competition assays: Preincubate antibody with immunizing peptide to confirm signal reduction. For example, ab72591 shows eliminated signal in both Western blot and immunofluorescence when preincubated with the immunizing peptide, confirming specificity .

  • Genetic models: Use HOXA11 knockdown/knockout samples as negative controls. The study in search result demonstrates HOXA11 knockdown using shRNA (TRCN0000413738 and TRCN0000417739) to validate antibody specificity.

  • Band size verification: HOXA11 has a predicted molecular weight of 34 kDa but may appear at approximately 30 kDa in Western blots due to post-translational modifications or protein processing .

  • Cross-validation: Compare results using antibodies targeting different epitopes or using orthogonal detection methods such as RNA expression analysis.

  • Epitope-tagged alleles: Consider using epitope-tagged alleles like the Hoxa11-3xflag model described in search result to overcome antibody limitations.

• What experimental approaches can be used to study HOXA11/HOXD11 genome-wide binding profiles?

  Recent methodological advances have overcome traditional limitations in studying HOX protein binding:

  • Epitope-tagged allele generation: The generation of Hoxa11-3xflag and Hoxd11-3xflag mouse models via CRISPR/Cas9 allows for precise detection without relying on antibody specificity . This approach addresses the historical challenges of:

    • Strong evolutionary conservation of the homeodomain

    • Generic AT-rich binding sites

    • Lack of specific antibodies

  • CUT&RUN analysis: Using FLAG-tagged HOX11 proteins with CUT&RUN (Cleavage Under Targets and Release Using Nuclease) methodologies has successfully demonstrated binding to known enhancers, such as the Six2 enhancer in developing kidney .

  • Conditional allele approaches: To study stage-specific binding, conditional alleles like the Hoxd11 conditional allele can be combined with epitope tagging for temporal control of expression .

  These approaches have validated that Hoxa11FLAG/FLAG; Hoxd11FLAG/FLAG animals are viable and fertile with no overt patterning defects, unlike Hoxa11/Hoxd11 mutants .

• What are the functional relationships between HOXA11 and other proteins in development and disease?

  HOXA11 functions through complex protein-protein interactions:

  • HOXA11-FOXO1A interaction: In placental mammals, HOXA11 underwent adaptive evolution to acquire the ability to interact with FOXO1A, enabling regulation of prolactin (PRL) expression in decidualized endometrial cells. This interaction is absent in non-placental species, as demonstrated by luciferase reporter assays .

  • HOXA11-Gli3 relationship: While HOXA11 regulates Gli3 expression transcriptionally in limb development, coimmunoprecipitation experiments using epitope-tagged Gli3 (3XFLAG-BirA-Gli3) showed no direct physical interaction between HOXA11 and Gli3 proteins .

  • HOXA11 in leukemia: HOXA11 expression is associated with improved response to cytarabine (Ara-C) in acute myeloid leukemia (AML) cells. Functional studies showed that HOXA11 knockdown reduced Ara-C sensitivity and apoptosis, while overexpression increased sensitivity .

  These interactions highlight how HOXA11 functions differently across developmental contexts and disease states.

• How can conditional genetic approaches be used to study HOXA11/HOXD11 functions in adult tissues?

  Conditional genetic models provide powerful tools for temporal control:

  • Conditional allele generation: A conditional Hoxd11 allele was generated by flanking exon 2 (containing the DNA-binding homeodomain) with loxP sites using CRISPR/Cas9 gene editing. This allows for temporal control of Hoxd11 deletion after normal development .

  • Validation approach: The functionality of conditional alleles can be validated by:

    1. PCR confirmation of recombination after Cre induction

    2. Demonstrating that early embryonic deletion recapitulates the conventional knockout phenotype

    3. Verifying protein loss through Western blot analysis

  • Application to adult functions: This approach revealed that Hox genes maintain critical roles in the adult skeleton by regulating differentiation of skeletal stem cells into the osteolineage, a function distinct from their embryonic patterning roles .

• What methodological considerations are important when using HOXA11/HOXD11 antibodies for cell-type specific analyses?

  For optimal cell-type specific analyses:

  • Dilution optimization: Different cell types may require different antibody dilutions. For example, immunofluorescence analysis of HUVEC cells using ab72591 works optimally at 1/500 dilution .

  • Fixation considerations: Crosslinking fixatives like paraformaldehyde may mask epitopes. Consider antigen retrieval methods when performing IHC or IF.

  • Background reduction: For immunofluorescence, include:

    1. Proper blocking with serum from the same species as the secondary antibody

    2. Negative controls omitting primary antibody

    3. Countervalidation using the immunizing peptide as a competitor

  • Marker co-localization: When studying specific cell populations, co-stain with established cell-type markers to confirm expression patterns.

• How do HOXA11 and HOXD11 proteins functionally interact in development, and how can researchers distinguish their individual contributions?

  HOXA11 and HOXD11 exhibit both redundant and unique functions:

  • Functional redundancy: Hoxa11 and Hoxd11 show partial redundancy in limb and kidney development. Single mutants have mild phenotypes, while double mutants exhibit severe defects in zeugopod (radius/ulna, tibia/fibula) formation and kidney development .

  • Experimental approaches to distinguish functions:

    1. Allelic series: Generate an allelic series (wildtype, single heterozygotes, double heterozygotes, single nulls, compound mutants) to assess dose-dependent effects .

    2. Paralog-specific knockdown: Use paralog-specific shRNAs or morpholinos with selective rescue as demonstrated for HOXA11 in search result .

    3. Epitope-tagged alleles: The Hoxa11-3xflag and Hoxd11-3xflag models allow separate tracking of each protein's binding and expression .

    4. Temporal-specific deletion: Use conditional alleles to delete each gene at different developmental stages to distinguish early versus late functions .

  These approaches have revealed that while both genes contribute to embryonic patterning, they may have distinct roles in adult tissue homeostasis and disease contexts.

• What are the critical technical considerations when using HOXA11/HOXD11 antibodies in Western blot analysis?

  For optimal Western blot results:

  • Protein extraction: Use appropriate lysis buffers that preserve protein integrity. RAW264.7 cell extracts have been successfully used at 5 μg per lane when studying HOXA11 .

  • Expected band size: Be aware that the observed band size (30 kDa) may differ from the predicted size (34 kDa) for HOXA11 in Western blots .

  • Antibody concentration: For HOXA11 antibody ab72591, a dilution of 1/500 provides optimal results . For dual-specificity HOXA11/HOXD11 antibodies, dilutions ranging from 1/500-1/2000 are recommended .

  • Controls: Include:

    1. Positive control lysates from cells known to express the target

    2. Competition with immunizing peptide

    3. Loading controls for normalization

  • Detection systems: Enhanced chemiluminescence (ECL) provides sufficient sensitivity for most applications, but fluorescent secondary antibodies may provide better quantitative results.

• How can researchers study the role of HOXA11/HOXD11 in disease processes like cancer and developmental disorders?

  Multiple methodological approaches have been employed:

  • Expression correlation studies: Meta-analysis using databases like Oncomine™ can identify correlations between HOXA11 expression and disease states or treatment responses. For example, higher HOXA11 expression correlates with better response to chemotherapy in AML .

  • Functional knockdown/overexpression: Modulating HOXA11 levels in disease models can reveal causative relationships. In AML cells, HOXA11 knockdown reduced sensitivity to Ara-C while overexpression increased sensitivity and apoptosis .

  • Mechanistic studies: Identify downstream targets by examining:

    1. Expression of apoptosis-related genes (NF-κB inhibitor α, transcription factor p65, transformation-related protein p53) following HOXA11 modulation

    2. Changes in cellular processes like differentiation, proliferation, and apoptosis

  • Evolutionary approaches: Examining adaptive evolution of HOXA11 across species has revealed how protein changes enabled novel functions, such as decidualization in placental mammals through interaction with FOXO1A .