HOXD9 Antibody, HRP conjugated

What is HOXD9 and why is it significant in research?

HOXD9 belongs to the homeobox family of genes, which encode a highly conserved family of transcription factors playing crucial roles in morphogenesis across multicellular organisms. The protein functions as a transcriptional regulator with various activation domains. HOXD9 is particularly significant in embryonic development and cellular differentiation, regulating downstream target genes essential for proper organ and limb formation . In mammals, HOXD9 is part of one of four homeobox gene clusters (HOXA, HOXB, HOXC, and HOXD) located on different chromosomes, with the HOXD genes specifically located at chromosome regions 2q31-2q37 . Deletions affecting the HOXD gene cluster have been associated with severe limb and genital abnormalities, highlighting its developmental importance . Additionally, HOXD9 has been implicated in pathological conditions, with significant roles in cancer progression including gastric cancer and gliomas .

What are the key characteristics of HOXD9 protein that researchers should know?

HOXD9 protein has several notable characteristics relevant to experimental design:

Researchers should note that while the calculated molecular weight is 36 kDa, the observed molecular weight in experimental conditions is typically 42 kDa, which may be due to post-translational modifications . This discrepancy is important to consider when validating antibody specificity and experimental results.

What types of HOXD9 antibodies are available and what is the significance of HRP conjugation?

Multiple types of HOXD9 antibodies are available for research purposes, including:

• Polyclonal rabbit antibodies (available from multiple vendors)

• Monoclonal mouse antibodies (including IgG1 kappa light chain variants)

• Various conjugated forms including HRP, PE, FITC, and Alexa Fluor® conjugates

• Specialized formulations such as ChIP-certified antibodies

HRP (horseradish peroxidase) conjugation is particularly significant for researchers because it eliminates the need for secondary antibody incubation in many applications. The direct conjugation provides several advantages:

• Reduced background signal due to fewer cross-reactivity issues

• Simplified experimental protocols with fewer washing steps

• Enhanced sensitivity in enzyme-linked detection systems

• Compatibility with various substrates including chemiluminescent, chromogenic, and fluorogenic options

• Greater reproducibility across experiments due to consistent antibody:enzyme ratios

The HRP conjugation is especially valuable for complex experimental designs where secondary antibody cross-reactivity could compromise results.

What are the validated applications for HOXD9 antibodies and their recommended dilutions?

HOXD9 antibodies have been validated for multiple experimental applications, with specific dilution recommendations varying by supplier and application:

When designing experiments, researchers should optimize these recommended dilutions for their specific experimental conditions and sample types. Initial titration experiments are advisable to determine optimal signal-to-noise ratios for each application. For Western blotting applications, 25 μg of total protein per lane has been shown to produce clear bands at the expected molecular weight using HOXD9 antibodies at 1:1000 dilution .

How should researchers optimize Western blotting protocols for HOXD9 detection?

Optimal Western blotting protocols for HOXD9 detection should include the following methodological considerations:

• Sample preparation:

  • Use appropriate lysis buffers that preserve protein integrity

  • Include protease inhibitors to prevent degradation

  • Load approximately 25 μg of total protein per lane

• Electrophoresis conditions:

  • Use gels that provide good resolution in the 35-45 kDa range to capture the 42 kDa HOXD9 protein

  • Include appropriate molecular weight markers

• Transfer considerations:

  • Optimize transfer time and voltage for proteins in the 42 kDa range

  • Verify transfer efficiency with reversible staining

• Blocking and antibody incubation:

  • Use 3% nonfat dry milk in TBST as blocking buffer

  • Dilute primary HOXD9 antibody to 1:1000 in blocking buffer

  • For HRP-conjugated antibodies, eliminate secondary antibody steps

  • For non-conjugated antibodies, use compatible secondary antibodies such as HRP-conjugated Goat anti-Rabbit IgG (H+L) at 1:10000 dilution

• Detection parameters:

  • Use appropriate ECL detection systems (e.g., ECL Basic Kit)

  • Optimize exposure times starting around 30 seconds

  • For weak signals, consider enhanced chemiluminescent substrates

Researchers should note that SH-SY5Y cells have been validated as a positive control for HOXD9 expression in Western blotting applications .

What methodologies should be followed when using HOXD9 antibodies for chromatin immunoprecipitation (ChIP)?

Chromatin immunoprecipitation using HOXD9 antibodies requires specialized methodologies:

• Antibody selection:

  • Use ChIP-certified HOXD9 antibodies

  • Verify species cross-reactivity with your experimental system

  • Consider polyclonal antibodies for broader epitope recognition

• Experimental design:

  • Include appropriate controls (normal IgG as negative control, known HOXD9 target as positive control)

  • Consider enrichment for HOXD9 binding sites in promoter regions

  • Design PCR primers for regions of interest based on predicted binding sites

• Protocol optimizations:

  • Optimize chromatin fragmentation to 200-500 bp fragments

  • Use sufficient antibody quantities for complete immunoprecipitation

  • Implement rigorous washing steps to reduce background

• Data analysis:

  • Perform PCR amplification to verify enrichment at suspected binding sites

  • Compare to control IgG pulldowns to confirm specificity

  • Consider quantitative PCR for precise enrichment measurements

This methodology has been successfully applied to identify HOXD9 binding sites, as demonstrated in studies of HOXD9 interaction with the PAXIP1-AS1 promoter region, where PCR amplification showed a band corresponding to 190 bp that included a specific binding site (-1503 to -1513) .

How can researchers validate the specificity of HOXD9 antibodies?

Validating antibody specificity is critical for generating reliable experimental data. For HOXD9 antibodies, consider these validation approaches:

• Multiple detection methods:

  • Compare results across different techniques (WB, IP, IF)

  • Verify expected molecular weight (42 kDa observed vs. 36 kDa calculated)

  • Confirm cellular localization pattern is consistent with nuclear transcription factor

• Positive and negative controls:

  • Use cell lines with known HOXD9 expression (e.g., SH-SY5Y)

  • Compare cancer vs. normal tissues (HOXD9 is differentially expressed in HeLa vs. normal cervical tissue)

  • Consider genetic approaches (siRNA knockdown, CRISPR knockout) to confirm specificity

• Epitope mapping:

  • Verify the immunogen sequence used for antibody generation (e.g., synthetic peptide corresponding to sequence within amino acids 100-200 of human HOXD9)

  • Consider peptide competition assays to confirm binding specificity

• Cross-reactivity assessment:

  • Check species cross-reactivity (human, monkey, mouse, rat depending on antibody)

  • Test for potential cross-reactivity with other HOX family members

• Reproducibility verification:

  • Compare results using antibodies from different suppliers or different lots

  • Validate consistency across experimental replicates

These validation steps are particularly important given the high sequence conservation among HOX family proteins and the potential for cross-reactivity.

What are the optimal storage and handling conditions for maintaining HOXD9 antibody activity?

Proper storage and handling of HOXD9 antibodies is essential for maintaining their activity and specificity over time:

• Storage temperature:

  • Store at -20°C for long-term preservation

  • Avoid repeated freeze-thaw cycles that can degrade antibody structure and activity

• Buffer composition:

  • Typical storage buffers contain PBS with preservatives and stabilizers (e.g., PBS with 0.05% proclin300, 50% glycerol, pH 7.3)

  • Do not alter or dilute storage buffers unless specified by the manufacturer

• Aliquoting recommendations:

  • Upon receipt, prepare single-use aliquots to minimize freeze-thaw cycles

  • Use sterile tubes and aseptic technique when handling antibodies

  • Document date of aliquoting and number of freeze-thaw cycles

• Working dilution handling:

  • Prepare fresh working dilutions on the day of experiment

  • Keep diluted antibodies cold (4°C) during experiment

  • Discard unused diluted antibody rather than storing

• Contamination prevention:

  • Use clean pipettes and tips when handling antibodies

  • Avoid bacterial or fungal contamination through proper laboratory technique

  • Monitor for signs of contamination (cloudiness, precipitates)

Following these guidelines will help maintain antibody performance and ensure reproducible experimental results.

What are common technical challenges when working with HOXD9 antibodies and how can they be addressed?

Researchers may encounter several technical challenges when working with HOXD9 antibodies:

• Non-specific binding:

  • Challenge: Background bands in Western blotting or non-specific staining in immunofluorescence

  • Solution: Optimize blocking conditions (3% nonfat dry milk in TBST has been validated) , increase washing stringency, and titrate antibody concentration

• Inconsistent signal intensity:

  • Challenge: Variable detection levels between experiments

  • Solution: Standardize protein loading (25 μg per lane recommended) , maintain consistent transfer conditions, and use internal loading controls

• Epitope masking:

  • Challenge: Reduced antibody binding due to protein modifications or interactions

  • Solution: Test multiple lysis conditions, consider native vs. denaturing conditions depending on application, and try antibodies targeting different epitopes

• Cross-reactivity with related HOX proteins:

  • Challenge: Difficulty distinguishing HOXD9 signal from other homeobox proteins

  • Solution: Use highly specific antibodies validated against multiple HOX proteins, include appropriate controls, and confirm results with orthogonal methods

• Detection sensitivity limitations:

  • Challenge: Weak signal when detecting endogenous HOXD9 in certain tissues

  • Solution: Optimize exposure times (starting with 30 seconds) , use enhanced chemiluminescent substrates for WB, and consider signal amplification methods for IF/IHC

These methodological adjustments can substantially improve experimental outcomes when working with HOXD9 antibodies.

How can HOXD9 antibodies be utilized to investigate transcriptional regulation mechanisms?

HOXD9 antibodies enable sophisticated investigations into transcriptional regulation mechanisms:

• Promoter binding studies:

  • Use ChIP assays to identify HOXD9 binding sites in promoter regions

  • As demonstrated in gastric cancer research, HOXD9 binding to the PAXIP1-AS1 promoter region can be detected through ChIP followed by PCR amplification

  • Combine with luciferase reporter assays to validate functional significance of binding sites

• Transcriptional complex analysis:

  • Employ co-immunoprecipitation with HOXD9 antibodies to identify protein interaction partners

  • Use sequential ChIP (re-ChIP) to identify co-occupancy of promoters with other transcription factors

  • Combine with mass spectrometry to identify novel HOXD9-interacting proteins

• Genome-wide binding profile:

  • Apply ChIP-seq methodologies using ChIP-certified HOXD9 antibodies

  • Integrate with RNA-seq data to correlate binding with gene expression changes

  • Analyze binding motifs to refine understanding of HOXD9 target specificity

• Epigenetic regulation mechanisms:

  • Investigate interactions between HOXD9 and chromatin modifiers

  • Explore the relationship between HOXD9 binding and histone modifications

  • Examine DNA methylation patterns at HOXD9 binding sites

Research has shown that HOXD9 acts as both activator and repressor, as evidenced by its overexpression resulting in downregulation of 54 lncRNAs and 495 protein-coding genes, while upregulating 11 lncRNAs and 229 protein-coding genes . These complex regulatory patterns can be further dissected using targeted approaches with specific HOXD9 antibodies.

What methodological approaches should be employed when studying HOXD9 in cancer progression and development?

Cancer researchers investigating HOXD9 should employ these methodological approaches:

• Expression analysis across cancer types:

  • Compare HOXD9 levels between tumor and matched normal tissues

  • Note differential expression patterns (e.g., present in HeLa cervical cancer cells but absent in normal cervical tissue)

  • Correlate expression with clinical parameters and patient outcomes

• Functional studies in cancer models:

  • Use HOXD9 antibodies to verify knockdown/overexpression efficiency

  • Examine effects on cell proliferation and survival (particularly relevant in gliomas)

  • Assess impact on epithelial-to-mesenchymal transition, which HOXD9 has been shown to induce

• Pathway analysis:

  • Investigate downstream effects of HOXD9 modulation on cancer-relevant pathways

  • Study regulation of specific targets like PAXIP1-AS1 in gastric cancer

  • Examine interaction with other oncogenic or tumor suppressor pathways

• Therapeutic targeting assessment:

  • Use HOXD9 antibodies to monitor protein levels following experimental therapies

  • Evaluate HOXD9 as a potential biomarker for treatment response

  • Investigate mechanisms of resistance related to HOXD9 expression

• In vivo tumor models:

  • Apply immunohistochemistry with HOXD9 antibodies to analyze expression in xenograft or transgenic models

  • Correlate with tumor progression metrics and treatment responses

  • Use tissue microarrays to assess expression across multiple tumor samples simultaneously

These approaches leverage the validated applications of HOXD9 antibodies while addressing the specific challenges of cancer research.

How can researchers integrate HOXD9 antibody data with genomic and transcriptomic analyses?

Integrating HOXD9 antibody-based protein data with genomic and transcriptomic analyses provides powerful insights:

• Multi-omics experimental design:

  • Perform parallel analyses on the same experimental system

  • Collect ChIP-seq data using ChIP-certified HOXD9 antibodies

  • Generate RNA-seq data to capture transcriptional changes

  • Add protein-level measurements via Western blotting or mass spectrometry

• Data integration methodologies:

  • Correlate HOXD9 binding sites (ChIP-seq) with expression changes (RNA-seq)

  • Map protein interaction networks (co-IP) to transcriptional networks

  • Develop computational models incorporating protein, DNA, and RNA data

• Causality testing:

  • Validate key findings through targeted experiments

  • Use site-directed mutagenesis of HOXD9 binding sites to confirm functional importance

  • Apply similar approaches as used in the PAXIP1-AS1 promoter study, where site-directed mutagenesis confirmed that the second HOXD9-binding site was critical for HOXD9-induced PAXIP1-AS1 transrepression

• Visualization and analysis tools:

  • Apply genome browsers to visualize ChIP-seq data alongside genomic features

  • Use pathway enrichment analyses to contextualize findings

  • Develop custom data visualization approaches for multi-omics integration

This integrated approach has revealed complex regulatory relationships, such as the finding that HOXD9 overexpression affects expression of both coding and non-coding genes, with 14 lncRNAs differentially expressed and annotated according to the HUGO Gene Nomenclature Committee database .

What are the considerations for using HOXD9 antibodies in developmental biology research?

Developmental biology research using HOXD9 antibodies requires specialized considerations:

• Temporal expression analysis:

  • Track HOXD9 expression across developmental stages

  • Consider the role of HOXD9 in early stages of joint development, where it is primarily expressed in articular cartilage

  • Correlate protein levels with developmental milestones

• Tissue-specific expression patterns:

  • Apply immunohistochemistry to map expression domains

  • Compare with in situ hybridization to distinguish transcriptional vs. post-transcriptional regulation

  • Consider regional differences in expression intensity and cellular localization

• Developmental perturbation models:

  • Use HOXD9 antibodies to validate knockout or knockdown models

  • Examine consequences of HOXD9 mutation or deletion on limb and genital development

  • Investigate compensatory mechanisms involving other HOX genes

• Evolutionary considerations:

  • Compare HOXD9 expression patterns across species

  • Examine conservation of regulatory mechanisms

  • Investigate species-specific functions and adaptations

• Technical adaptations for developmental samples:

  • Optimize fixation protocols for embryonic tissues

  • Adjust antibody concentrations for developmental samples

  • Consider whole-mount immunostaining approaches for spatial analysis

The comprehensive understanding of HOXD9 in development has implications for both basic developmental biology and for understanding congenital abnormalities associated with HOXD gene cluster disruptions .

How should researchers approach the study of HOXD9 in inflammatory and degenerative conditions?

Emerging evidence suggests HOXD9 involvement in inflammatory and degenerative conditions, requiring specialized research approaches:

• Expression analysis in disease models:

  • Compare HOXD9 levels in normal vs. pathological tissues

  • Note the elevated expression in synovial tissue of arthritic mice compared to normal mice

  • Correlate expression with disease progression and severity

• Inflammatory response studies:

  • Examine relationships between inflammatory mediators and HOXD9 expression

  • Investigate potential feedback loops in chronic inflammation

  • Study effects of anti-inflammatory interventions on HOXD9 levels

• Cellular specificity determination:

  • Use immunofluorescence with cell-type specific markers to identify HOXD9-expressing cells

  • Compare expression patterns in immune cells vs. tissue-resident cells

  • Investigate cell-specific functions through conditional approaches

• Signaling pathway integration:

  • Map interactions between inflammatory signaling cascades and HOXD9 regulation

  • Investigate post-translational modifications affecting HOXD9 function under inflammatory conditions

  • Explore potential therapeutic targets within these pathways

• Translational considerations:

  • Develop standardized protocols for analyzing HOXD9 in clinical samples

  • Investigate HOXD9 as a potential biomarker for disease activity

  • Consider HOXD9-targeting approaches for therapeutic development

These methodological approaches acknowledge HOXD9's emerging role in inflammatory conditions while addressing the technical challenges of studying transcription factors in complex disease environments.