HOXD1 Antibody, HRP conjugated

What is HOXD1 and why is it a significant research target?

HOXD1 belongs to the Antp homeobox family and functions as a nuclear protein with a homeobox DNA-binding domain. Research indicates HOXD1 plays crucial roles in differentiation and limb development, with mutations associated with severe developmental defects on the anterior-posterior limb axis . HOXD1 has emerged as an important research target due to its involvement in:

• Embryonic development and patterning

• Transcriptional regulation of developmental processes

• Cancer progression and prognosis across multiple cancer types

• Oligodendroglial cell development and myelination

• Neural circuit formation in nociceptors

HOXD1 has been implicated in both developmental disorders and cancer biology, making antibodies against this protein valuable tools for investigating developmental pathways and disease mechanisms .

What applications are validated for HOXD1 antibody, HRP conjugated?

HOXD1 antibodies, including HRP-conjugated versions, have been validated for several applications:

• Enzyme-Linked Immunosorbent Assay (ELISA): HRP-conjugated HOXD1 antibodies are particularly valuable for direct ELISA applications with dilutions ranging from 1:2000 to 1:10000

• Western Blotting (WB): Used for detecting HOXD1 protein in tissue and cell lysates, often at dilutions between 1:500-1:5000

• Immunohistochemistry (IHC): For detecting HOXD1 in tissue sections at dilutions of 1:500-1:1000

These applications allow researchers to detect and analyze HOXD1 expression in various experimental contexts, from protein expression analysis to localization studies in tissues.

How does HRP conjugation enhance antibody functionality for HOXD1 detection?

HRP (Horseradish Peroxidase) conjugation provides several advantages for HOXD1 detection:

• Direct detection without secondary antibodies, simplifying experimental workflows

• Signal amplification for increased sensitivity, allowing detection of low abundance HOXD1

• Compatibility with multiple detection substrates (colorimetric, chemiluminescent)

• Stability in various buffers and experimental conditions

The enzymatic activity of HRP enables conversion of substrate molecules into detectable signals, thereby amplifying the detection of even small amounts of HOXD1 protein. This is particularly valuable when studying HOXD1 in biological samples where expression may be limited .

What is the enhanced lyophilization method for HRP-HOXD1 antibody conjugation?

Research has demonstrated that incorporating lyophilization into the conjugation protocol significantly enhances antibody sensitivity. The enhanced labeling procedure involves:

• Activation of HRP using 0.15 M Sodium metaperiodate

• Desalting by dialysis with 1× PBS for 3 hours at room temperature

• Freezing activated HRP at -80°C for 5-6 hours

• Overnight lyophilization of the frozen HRP

• Mixing lyophilized HRP with HOXD1 antibody (1:4 molar ratio of antibody:HRP)

• Incubation at 37°C for 1 hour in a thermomixer

• Addition of sodium cyanoborohydride (1/10th volume) for Schiff's base reaction

• Incubation at 4°C for 2 hours followed by overnight dialysis against 1× PBS

This modified protocol has demonstrated significantly improved sensitivity compared to classical conjugation methods:

The p-value between methods was highly significant (p<0.001), demonstrating that lyophilization substantially enhances the conjugation efficiency and resulting antibody performance .

How should researchers optimize HOXD1 antibody, HRP conjugated dilutions for various applications?

Optimal dilution determination requires systematic titration experiments for each application:

For ELISA:

• Starting recommended range: 1:2000-1:10000

• Perform checkerboard titration with 2-fold serial dilutions

• Include appropriate positive and negative controls

• Select dilution with optimal signal-to-noise ratio

For Western Blotting:

• Starting recommended range: 1:500-1:5000

• Test multiple dilutions on the same membrane if possible

• Evaluate background signal and specific band intensity

• Optimize incubation time and temperature (typically 1-2 hours at room temperature or overnight at 4°C)

For Immunohistochemistry:

• Starting recommended range: 1:500-1:1000

• Test on known positive and negative control tissues

• Evaluate specificity and background staining

• Optimize antigen retrieval methods if necessary

The antibody storage buffer (typically 50% glycerol, 0.01M PBS, pH 7.4 with 0.03% Proclin 300) should be considered when calculating dilution factors .

What controls are essential when using HOXD1 antibody, HRP conjugated in research?

Comprehensive experimental design should include multiple levels of controls:

Negative Controls:

• Isotype controls (rabbit polyclonal IgG for many HOXD1 antibodies)

• Secondary antibody-only controls (for indirect detection methods)

• Bovine serum albumin (BSA) as a binding control in protein-DNA interaction studies

• Tissues or cells known not to express HOXD1

Positive Controls:

• Recombinant HOXD1 protein

• Cell lines with confirmed HOXD1 expression (e.g., RT-4, U-251 MG cells)

• Tissues with established HOXD1 expression (e.g., brain tissue, lung tissue)

Specificity Controls:

• Competitive binding with excess unlabeled antibody

• Pre-adsorption of antibody with immunizing peptide

• Multiple antibodies targeting different epitopes of HOXD1

• For DNA binding studies, competition assays with specific and mutated probes

The search results specifically mention using rat lung tissue, rat kidney tissue, and mouse lung tissue as positive controls for Western blot validation of HOXD1 antibodies .

What species reactivity is documented for HOXD1 antibodies?

HOXD1 antibodies exhibit varying cross-reactivity profiles depending on the immunogen and manufacturing process. According to the search results, HOXD1 antibodies have been validated with the following reactivity profile:

When selecting a HOXD1 antibody for cross-species applications, researchers should consider the percent identity of the target epitope. For example, one antibody targeting the C-terminal region demonstrated predicted reactivity with cow (90%), dog (100%), guinea pig (92%), horse (90%), and zebrafish (90%) .

How do interspecies differences in HOXD1 expression affect experimental design?

Studies have revealed significant species-specific differences in HOXD1 expression patterns that researchers must consider:

• In mice, HOXD1 is expressed primarily in mesoderm and neural crest cells

• In Xenopus, HOXD1 shows a wider expression pattern including future hindbrain and associated neural crest

• Expression timing and patterns differ across species during development

These differences could substantially impact experimental interpretation when studying HOXD1 across species. For example, research has demonstrated that in Xenopus, knockdown of Hox paralogous group 1 genes (including HOXD1) affects hindbrain patterning and neural crest migration into pharyngeal arches, with effects more severe than predicted from single and double knockouts in other organisms .

When designing comparative studies, researchers should:

• Characterize baseline HOXD1 expression in each species at relevant developmental timepoints

• Consider potential functional redundancy with other HOX genes

• Select appropriate developmental stages for cross-species comparisons

• Use species-specific positive controls to validate antibody reactivity

How can HOXD1 antibodies be used to study cancer progression and prognosis?

HOXD1 has emerged as a significant biomarker in multiple cancer types, with expression patterns correlating with clinical outcomes. A pan-cancer analysis revealed critical associations between HOXD1 expression and patient prognosis:

Methodological approaches for studying HOXD1 in cancer include:

• Immunohistochemical analysis of tumor tissue microarrays to correlate HOXD1 expression with histopathological features

• Western blot and ELISA quantification of HOXD1 in tumor samples versus normal tissues

• Correlation of HOXD1 protein levels with clinical stage and histological grade

• Integration of HOXD1 expression data with survival analysis

• In vitro functional studies using HOXD1 overexpression or knockdown in cancer cell lines

Recent research has demonstrated that HOXD1 inhibits lung adenocarcinoma progression and is regulated by DNA methylation, suggesting potential therapeutic implications .

What is the relationship between HOXD1 and DNA methylation in cancer development?

Recent studies have revealed a critical regulatory relationship between DNA methylation and HOXD1 expression in cancer:

• DNA hypermethylation occurs in the promoter region of HOXD1 in lung adenocarcinoma

• This hypermethylation is associated with DNA methyltransferase activity

• Methylation status correlates with HOXD1 expression levels

• As a transcription factor, HOXD1 regulates downstream target genes including BMP2 and BMP6

To investigate this relationship, researchers can employ:

• Targeted bisulfite sequencing to analyze methylation patterns in the HOXD1 promoter

• Chromatin immunoprecipitation assays to study DNA methyltransferase binding

• HOXD1 antibodies in combination with methylation analysis to correlate protein expression with epigenetic status

• Transcriptional analysis of BMP2/BMP6 following HOXD1 overexpression or knockdown

These findings suggest that epigenetic regulation of HOXD1 may play a significant role in cancer development, with HOXD1 serving as both a target of epigenetic regulation and a mediator of downstream effects on target genes .

How can researchers determine HOXD1 binding specificity to DNA sequences?

HOXD1 functions as a sequence-specific transcription factor, and characterizing its DNA binding properties is crucial for understanding its biological function. Research has established methods to determine binding specificity:

• Electrophoretic Mobility Shift Assays (EMSAs): Studies have used EMSAs to determine the binding parameters of HOXD1 to target sequences. The dissociation coefficient constant (Kᴅ) of the HOXD1-MOG complex was measured at 1.9 × 10⁻⁷ M, with a dissociation rate constant (kd) of 1.3 × 10⁻³ s⁻¹, resulting in a half-life (t₁/₂) of 15 minutes .

• Competition Assays: These can be performed using:

  • Wild-type probe

  • Mutant probes with specific modifications in the binding sequence

  • Varying concentrations (0, 5×, 50×, 150×) of cold probe to hot normal probe

• Mutational Analysis: Research identified that the TAATTG core of the binding sequence is critical for HOXD1 specificity. Mutations in this core sequence (changing TAAT to TACT or TAATTG to TAATCC) severely affected binding affinity, while mutations in adjacent sites had less impact .

The consensus binding sequence for HOXD1 was identified as TTTAATTGTA, although neighboring TAAT sites may also contribute to binding .

What is the role of HOXD1 in oligodendrocyte development and myelin formation?

Research has identified HOXD1 as a regulator of oligodendrocyte development through its interaction with myelin-related genes:

• HOXD1 is expressed throughout oligodendrocyte development, as demonstrated by immunocytochemical analysis of primary mixed glial cultures

• The human myelin protein gene, myelin oligodendrocyte glycoprotein (MOG), was identified as a putative downstream target of HOXD1

• HOXD1 binds to a specific sequence in the MOG promoter region

• This binding appears to regulate MOG expression during oligodendrocyte development

To study this relationship, researchers can use:

• Double immunolabeling with HOXD1 antibodies and oligodendrocyte markers (A2B5, O4, GalC, MBP)

• Chromatin immunoprecipitation to identify HOXD1 binding sites in myelin-related gene promoters

• Reporter gene assays to measure transcriptional activity

• Functional studies using HOXD1 overexpression or knockdown in oligodendrocyte precursor cells

These findings suggest HOXD1 may play a role in regulating myelination, with potential implications for demyelinating disorders and nervous system development .

What storage conditions are recommended to maintain HRP-conjugated HOXD1 antibody activity?

Proper storage is critical for maintaining the activity of HRP-conjugated HOXD1 antibodies:

Long-term storage:

• Store at -20°C or -80°C

• Avoid repeated freeze-thaw cycles

• Store in small aliquots to prevent freeze-thaw damage

Short-term storage (up to 1 week):

• Store at 2-8°C

Buffer composition:

• Typical storage buffer: 50% glycerol, 0.01M PBS, pH 7.4

• Preservative: 0.03% Proclin 300

• Some formulations may contain small amounts of sucrose (2%) as a stabilizer

When handling the antibody:

• Avoid unnecessary exposure to light

• Return to recommended storage conditions promptly after use

• Allow frozen antibody to thaw completely at room temperature before use

• Gently mix by inversion; avoid vigorous vortexing

Proper storage significantly impacts antibody performance, particularly for conjugated antibodies where both the antibody specificity and enzymatic activity of HRP must be preserved .

How can researchers verify the activity and specificity of HOXD1 antibody, HRP conjugated?

Multiple approaches should be used to verify both the immunological specificity and enzymatic activity of HRP-conjugated HOXD1 antibodies:

Specificity Verification:

• Western Blot Analysis:

  • Confirm detection of a band at the expected molecular weight (34 kDa for HOXD1)

  • Test against tissue lysates with known HOXD1 expression (e.g., rat lung, rat kidney, mouse lung)

  • Include negative controls (tissues not expressing HOXD1)

• ELISA Validation:

  • Establish dose-response curves using recombinant HOXD1 protein

  • Determine lower limit of detection (research indicates sensitivity as low as 1.5 ng with enhanced conjugation methods)

  • Compare signal between positive and negative samples

HRP Activity Verification:

• Spectrophotometric Analysis:

  • UV-spectroscopy wavelength scan (280-800 nm range)

  • HOXD1 antibody typically shows peak at 280 nm

  • HRP shows characteristic peak at 430 nm

  • Conjugated antibody should show both peaks with potential shift at 430 nm

• SDS-PAGE Analysis:

  • Compare migration patterns of conjugated vs. unconjugated antibody

  • Properly conjugated antibody-HRP complex will show reduced mobility

• Functional Testing:

  • Test substrate conversion (TMB, DAB, or chemiluminescent substrates)

  • Compare signal strength between freshly prepared conjugate and stored conjugate

  • Establish activity retention over time under different storage conditions

How are HOXD1 antibodies being used to study developmental processes and neural circuits?

Recent research has revealed critical roles for HOXD1 in developmental processes, particularly in neural circuit formation:

• Nociceptor Development:

  • HOXD1 expression is regulated by NGF/TrkA signaling in mouse nociceptors

  • HOXD1 instructs development of mammal-specific features of nociceptive neural circuitry

  • Genetic manipulations demonstrate HOXD1's role in behavioral sensitivity to extreme cold

• Species-Specific Neural Development:

  • Differential expression of HOXD1 across vertebrate species contributes to species-specific features of nociceptor circuits

  • In mice lacking HOXD1, nociceptor circuitry resembles that found in chicks

  • Ectopic expression of HOXD1 in developing chick nociceptors promotes mouse-like axonal projections

Researchers can use HOXD1 antibodies to:

• Track HOXD1 expression during critical developmental periods

• Correlate HOXD1 expression with specific neuronal phenotypes

• Study interspecies differences in neural development

• Investigate the relationship between HOXD1 and other developmental regulators

These studies suggest HOXD1 represents a rapidly evolving component of developmental signaling pathways that contribute to species-specific neural circuit formation, with implications for evolutionary biology and neurodevelopment .

What methodological approaches can be used to study HOXD1's transcriptional regulatory networks?

As a transcription factor, HOXD1 regulates multiple downstream targets. Several methodological approaches can be employed to elucidate these regulatory networks:

• Chromatin Immunoprecipitation (ChIP):

  • Use HOXD1 antibodies to immunoprecipitate HOXD1-bound chromatin

  • Combine with sequencing (ChIP-seq) to identify genome-wide binding sites

  • Verify binding sites using EMSA with synthetic oligonucleotides

• Reporter Gene Assays:

  • Clone promoter regions of putative target genes into reporter constructs

  • Measure transcriptional activity in response to HOXD1 overexpression or knockdown

  • Mutate binding sites to confirm direct regulation

• Expression Profiling:

  • Perform RNA-seq following HOXD1 manipulation (overexpression, knockdown)

  • Identify differentially expressed genes as potential targets

  • Validate with qRT-PCR and protein-level analysis

• Integrative Analysis:

  • Combine ChIP-seq data with expression profiles to identify direct targets

  • Perform pathway analysis to identify enriched biological processes

  • Construct regulatory networks from integrated datasets

Recent research has identified several HOXD1 targets, including:

• BMP2 and BMP6, with HOXD1 serving as a transcriptional activator

• MOG (myelin oligodendrocyte glycoprotein), suggesting a role in myelination

• Genes involved in anterior-posterior axis patterning

These methodological approaches provide a framework for investigating the complex regulatory networks controlled by HOXD1 in different biological contexts .