HOXD13 Antibody

What are the key characteristics of available HOXD13 antibodies?

HOXD13 antibodies are available in multiple formats with varying host species and applications. The primary types currently available include rabbit polyclonal antibodies (such as 23520-1-AP) and goat polyclonal antibodies (such as STJ72144) . These antibodies target different regions of the HOXD13 protein, including internal regions or specific fusion proteins used as immunogens. When selecting an antibody, researchers should consider several critical parameters:

The choice between these antibodies should be guided by your specific application, target species, and experimental design requirements .

What are the recommended applications and dilutions for HOXD13 antibodies?

HOXD13 antibodies can be utilized across several experimental applications with specific dilution recommendations to achieve optimal results. When designing your experiments, consider the following application-specific guidelines:

It is important to note that these dilutions serve as starting points, and researchers should optimize conditions for their specific experimental systems. For example, the rabbit polyclonal antibody ab229234 has been successfully used at a 1:1000 dilution for Western blot in Jurkat cell lysates . When working with new cell lines or tissues, a titration experiment is recommended to determine the optimal antibody concentration that provides the best signal-to-noise ratio .

How should HOXD13 antibodies be stored and handled for maximum stability?

Proper storage and handling of HOXD13 antibodies is crucial for maintaining their performance and extending their shelf life. Based on manufacturer recommendations, observe the following guidelines:

For the rabbit polyclonal antibody (23520-1-AP):

• Store at -20°C

• Stable for one year after shipment

• Aliquoting is unnecessary for -20°C storage

• The 20μl size contains 0.1% BSA as a stabilizer

For the goat polyclonal antibody (STJ72144):

• Store at -20°C upon receipt

• Minimize freeze-thaw cycles

• Formulated in Tris saline with 0.02% sodium azide, pH 7.3, and 0.5% bovine serum albumin

To maximize antibody stability, avoid repeated freeze-thaw cycles by preparing working aliquots. When using the antibody, thaw aliquots completely before use and keep on ice during handling. Return to -20°C promptly after use to prevent degradation. The presence of glycerol, BSA, and sodium azide in the formulations helps maintain stability, but researchers should still follow good laboratory practices for antibody handling .

What are the optimal protocols for HOXD13 detection in Western blot applications?

Western blot detection of HOXD13 requires specific optimizations to ensure successful protein visualization. Based on experimental data from published protocols, the following methodological approach is recommended:

• Sample Preparation:

  • For cell lines such as HL-60 or A549 cells, use standard lysis buffers containing protease inhibitors

  • Include phosphatase inhibitors if phosphorylation states are relevant

  • Load 20-30 μg of total protein per lane (30 μg was used successfully for Jurkat cell lysates)

• Electrophoresis Conditions:

  • Use 10% SDS-PAGE gels for optimal separation

  • Run at 100-120V until sufficient separation is achieved

• Transfer and Antibody Incubation:

  • Transfer proteins to PVDF or nitrocellulose membranes

  • Block with 5% non-fat milk or BSA in TBST

  • Incubate with primary antibody (1:200-1:1000 dilution) overnight at 4°C

  • Wash thoroughly with TBST (3-5 times, 5 minutes each)

  • Incubate with appropriate HRP-conjugated secondary antibody

  • Visualize using enhanced chemiluminescence

• Controls and Validation:

  • Include positive controls (A549 or HL-60 cell lysates have shown positive results)

  • Include loading controls (β-actin or NFY-B have been used successfully)

  • Expect band detection at approximately 36 kDa, which corresponds to the calculated molecular weight of HOXD13

When troubleshooting Western blots, consider that the observed molecular weight of HOXD13 is 36 kDa, matching its calculated weight of 343 amino acids. Deviations from this size may indicate post-translational modifications, proteolytic processing, or non-specific binding .

How can HOXD13 function be studied through DNA binding assays?

Investigating HOXD13's function as a DNA-binding transcription factor requires specialized techniques to analyze its interaction with target DNA sequences. Based on published methodologies, the following approaches are recommended:

• Electrophoretic Mobility Shift Assays (EMSA):

  • Produce HOXD13 protein in reticulocyte lysates or recombinant systems

  • Use 32P-labeled oligonucleotides containing the TTACGAG HOXD13 binding site as a probe

  • Perform binding reactions according to standard protocols

  • Run on non-denaturing polyacrylamide gels to separate protein-DNA complexes

  • Include specificity controls such as unlabeled competitor oligonucleotides

• Chromatin Immunoprecipitation (ChIP):

  • Cross-link protein-DNA complexes in living cells (HEK293 or SW1353 cells have been used)

  • Isolate and shear chromatin

  • Immunoprecipitate with anti-HOXD13 antibodies

  • Purify DNA and analyze by PCR or sequencing

  • This approach has successfully demonstrated HOXD13 binding to characterized human DNA replication origins

• Protein-Protein Interaction Analysis:

  • Investigate HOXD13 interactions with replication machinery components

  • Use coimmunoprecipitation assays with antibodies against HA-tagged HOXD13 or interaction partners

  • GST pulldown assays can be performed using bacterially produced GST-HOXD13HD fusion protein

  • In vitro transcription/translation can be used to produce 35S-labeled proteins for interaction studies

These methodologies have revealed that HOXD13 can interact with components of the DNA replication machinery, including CDC6 and ORC2, suggesting its role in DNA replication origin function beyond transcriptional regulation .

What approaches are effective for studying HOXD13 cellular localization?

HOXD13 functions as a transcription factor with predominantly nuclear localization. Effective visualization and analysis of its cellular distribution require specific immunofluorescence techniques:

• Immunofluorescence (IF) Optimization:

  • Use A549 cells as a positive control system

  • Fix cells with 4% paraformaldehyde or methanol

  • Permeabilize with 0.1-0.5% Triton X-100

  • Block with appropriate serum (5-10%)

  • Incubate with HOXD13 antibody at 1:50-1:500 dilution

  • Use fluorophore-conjugated secondary antibodies

  • Counterstain nuclei with DAPI or Hoechst

• Immunohistochemistry (IHC) Applications:

  • The goat polyclonal antibody (STJ72144) has been validated for paraffin-embedded human prostate tissue

  • Use at 1-2 μg/ml concentration

  • Expected result: nuclear staining in secretory cells

  • Include appropriate positive and negative controls

• Subcellular Fractionation:

  • Separate nuclear and cytoplasmic fractions

  • Confirm fractionation efficiency with compartment-specific markers

  • Analyze HOXD13 distribution by Western blot

  • Nuclear extracts from HEK and HEK-LD13IΔN cells have been used successfully

• Live Cell Imaging Considerations:

  • For dynamic studies, tagged versions of HOXD13 may be necessary

  • Validate that tags do not interfere with normal localization

  • TO-PRO3 vital dye can be used for nuclear counterstaining in live cells

When interpreting results, remember that HOXD13 exhibits strong nuclear localization, consistent with its function as a transcription factor. Any significant cytoplasmic staining should be carefully validated to rule out antibody cross-reactivity or experimental artifacts .

How can researchers validate HOXD13 antibody specificity?

Antibody validation is critical for ensuring experimental reproducibility and accurate data interpretation. For HOXD13 antibodies, implement the following comprehensive validation strategy:

• Genetic Approaches:

  • Use HOXD13 knockout or knockdown models (siRNA has been used successfully in HEK-LD13IΔN cells)

  • Compare antibody reactivity between wild-type and HOXD13-depleted samples

  • Verify knockdown efficiency by independent methods (qPCR, alternative antibodies)

• Overexpression Systems:

  • Express tagged versions of HOXD13 (Flag-HOXD13 or HA-HOXD13)

  • Confirm co-localization of anti-tag and anti-HOXD13 antibody signals

  • This approach has been utilized in SW1353 and HEK293 expression systems

• Peptide Competition:

  • Pre-incubate antibody with immunizing peptide (e.g., KSSFPGDVALNQPD for STJ72144)

  • Compare signal between blocked and unblocked antibody

  • Specific signals should be eliminated by peptide competition

• Cross-Reactivity Assessment:

  • Test antibody against related HOX proteins

  • For murine models, confirm specificity against homologous genes (Hoxa2, Hoxa10, Hoxa11, Hoxb9, Hoxb13, Hoxc8, Hoxc11, Hoxd9)

  • PCR-Sanger sequencing can be used to identify potential cross-reactivity

• Multi-Application Consistency:

  • Verify consistent results across different applications (WB, IF, IHC)

  • Compare reactivity patterns in different cell types with known HOXD13 expression profiles

Documented validation data shows that rabbit polyclonal antibody 23520-1-AP successfully detects HOXD13 in HL-60 and A549 cells by Western blot, and in A549 cells by immunofluorescence, while the goat polyclonal antibody STJ72144 shows nuclear staining in secretory cells of human prostate by IHC .

What are the key considerations for cross-species reactivity in HOXD13 antibodies?

When designing experiments involving multiple species or animal models, understanding the cross-species reactivity of HOXD13 antibodies is essential:

To ensure reliable cross-species application:

• Sequence Homology Analysis:

  • Analyze the conservation of the immunogen sequence across target species

  • For STJ72144, verify conservation of the KSSFPGDVALNQPD epitope

  • Higher sequence homology increases the likelihood of cross-reactivity

• Positive Control Selection:

  • Include tissues or cell lines with known HOXD13 expression from each species

  • For mouse studies, embryonic limb buds at E11.0-E11.5 show HOXD13 expression

  • For human studies, prostate tissue shows nuclear HOXD13 staining in secretory cells

• Validation Experiments:

  • Perform Western blot with positive control samples from each species

  • Verify expected molecular weight (may vary slightly between species)

  • For novel applications, validate with additional techniques like RT-PCR or RNA-seq

• Species-Specific Optimizations:

  • Adjust antibody concentrations and incubation times for each species

  • Modify blocking conditions to reduce background in specific tissues

  • Select appropriate negative controls for each species

When studying mutant models such as the Hoxd13Q50R mouse model, confirm that mutations do not affect antibody epitope recognition. Techniques such as PCR-Sanger sequencing have been used to verify mutations in exon 2 of Hoxd13 and related genes, ensuring that antibody detection remains reliable in mutant contexts .

How can researchers optimize HOXD13 chromatin immunoprecipitation (ChIP) experiments?

ChIP is a powerful technique for studying HOXD13's genomic binding sites. Based on published protocols, the following optimizations are recommended:

• Cell Line Selection:

  • HEK293 human embryonic kidney cells and SW1353 human humeral bone chondroblast cells have been successfully used

  • Consider cell types relevant to your research question (e.g., limb development may require embryonic or developmental cell models)

• Crosslinking Optimization:

  • Standard formaldehyde crosslinking (typically 1% for 10 minutes)

  • Optimize crosslinking time based on cell type and antibody accessibility

  • Include appropriate controls (input, IgG, positive control antibody)

• Sonication Parameters:

  • Optimize sonication conditions to achieve chromatin fragments of 200-500 bp

  • Verify fragment size by agarose gel electrophoresis

  • Insufficient fragmentation can reduce immunoprecipitation efficiency

• Antibody Selection:

  • Use ChIP-validated antibodies when available

  • For tagged HOXD13 constructs, anti-tag antibodies (anti-HA, anti-Flag) may provide higher specificity

  • Include appropriate negative controls with IgG from the same species

• Data Analysis:

  • Normalize to input samples

  • Include genomic regions known not to bind HOXD13 as negative controls

  • For genome-wide studies, consider ChIP-seq with appropriate bioinformatic pipelines

HOXD13 ChIP experiments have successfully demonstrated its binding to characterized human DNA replication origins, revealing a novel function beyond its classical role as a transcription factor. This suggests that when designing ChIP experiments, researchers should consider both promoter regions of target genes and potential DNA replication origins .

What strategies are effective for investigating HOXD13 mutations in experimental models?

Studying HOXD13 mutations requires specialized genetic approaches, particularly for modeling human disorders such as Syndactyly Type V (SDTY5):

• TALE-Mediated Mutagenesis:

  • Design TALEs targeting specific exons (second exon of murine Hoxd13 has been targeted)

  • Include donor DNA templates with desired mutations (e.g., A-to-G mutation at position 1769)

  • Consider incorporating restriction sites (e.g., NdeI) to facilitate genotyping

  • For synonymous changes, ensure they don't affect splicing or expression

• Genotyping Strategies:

  • Design PCR primers flanking the mutation site

  • For mutations introducing restriction sites, use restriction enzyme digestion (e.g., NdeI)

  • Confirm mutations by Sanger sequencing

  • Check for off-target effects in homologous genes

• Phenotypic Analysis:

  • For limb development studies, examine embryos at critical developmental stages (E11.0-E11.5)

  • Use whole-mount in situ hybridization (WISH) to analyze expression patterns of HOXD13 targets (e.g., Bmp2)

  • Document and quantify phenotypic changes between wild-type and mutant models

• Molecular Characterization:

  • Assess changes in target gene expression (e.g., downregulation of Bmp2 expression)

  • Analyze potential ectopic expression patterns

  • Compare expression at multiple developmental stages to identify temporal abnormalities

Research on the Hoxd13Q50R mutation demonstrates how these approaches can reveal pathogenic mechanisms, showing both downregulation and ectopic expression of downstream targets like Bmp2. This underscores the importance of examining both spatial and temporal expression patterns when characterizing HOXD13 mutations .

How can researchers investigate HOXD13 protein-protein interactions?

Understanding HOXD13's interaction network is crucial for deciphering its molecular functions beyond DNA binding. The following methodologies have proven effective:

• Co-Immunoprecipitation (Co-IP):

  • Use total or nuclear extracts from cells expressing HA-HOXD13 and potential interaction partners

  • Immunoprecipitate with anti-HA, anti-HOXD13, or antibodies against predicted interaction partners

  • Western blot to detect co-precipitated proteins

  • This approach has revealed interactions between HOXD13 and replication machinery components (geminin, CDC6, Orc2)

• GST Pulldown Assays:

  • Produce GST-HOXD13HD fusion protein in bacterial systems

  • Immobilize on glutathione-Sepharose 4B resin

  • Challenge with in vitro-transcribed/translated 35S-labeled potential interaction partners

  • This method has been used successfully with CDC6 and Orc2

• In Vitro Binding Assays:

  • Produce HOXD13 and potential interaction partners in reticulocyte lysates

  • Perform binding reactions according to standard protocols

  • Analyze by SDS-PAGE or non-denaturing electrophoresis

  • Include appropriate controls to verify specificity

• Quantitative Analysis:

  • When possible, perform quantitative binding assays to determine binding affinities

  • Include competition experiments with unlabeled proteins

  • Consider the effects of post-translational modifications on interaction strength

These approaches have revealed unexpected interactions between HOXD13 and components of the DNA replication machinery, suggesting roles beyond transcriptional regulation. This underscores the importance of unbiased screening approaches when investigating protein-protein interactions .