hoxb5a Antibody

What is HOXB5/hoxb5a and why is it significant for developmental biology research?

HOXB5 (in humans) or hoxb5a/hoxb5b (in zebrafish) is a sequence-specific transcription factor that functions as part of a developmental regulatory system providing cells with specific positional identities along the anterior-posterior axis . It plays critical roles in various developmental processes, including foregut endoderm morphogenesis in zebrafish , motor neuron diversification , and hematopoietic stem cell regulation . Its evolutionary conservation across species makes it valuable for comparative developmental studies.

What types of HOXB5/hoxb5a antibodies are currently available for research?

Several types of antibodies targeting HOXB5/hoxb5a are available:

• Polyclonal antibodies: Typically raised in rabbits against specific epitopes, such as N-terminal regions (amino acids 1-30) in mouse Hoxb5

• Monoclonal antibodies: Including rabbit monoclonal antibodies that offer high specificity

• Species-specific antibodies: Antibodies validated for human, mouse, or zebrafish HOXB5/hoxb5 proteins

These antibodies have been validated for various applications including Western blotting (WB), immunocytochemistry/immunofluorescence (ICC/IF), and flow cytometry .

How should I validate a HOXB5/hoxb5a antibody for my specific experimental system?

Validation should include multiple complementary approaches:

• Positive and negative controls:

  • Use tissue or cell lines known to express HOXB5 (e.g., fetal lung, fetal kidney, or U-87 MG cells for human HOXB5)

  • Include knockout or knockdown samples where possible

• Cross-validation with multiple techniques:

  • Western blot to confirm antibody recognizes a protein of the expected size (~29 kDa)

  • Immunofluorescence to confirm expected subcellular localization (nuclear)

  • RT-PCR to correlate protein detection with mRNA expression

• Peptide competition assay:

  • Pre-incubate antibody with the immunizing peptide to demonstrate binding specificity

What is the recommended protocol for immunofluorescence staining using HOXB5/hoxb5a antibodies?

Based on validated protocols :

• Fixation and permeabilization:

  • Fix cells with 4% paraformaldehyde (PFA) for 15 minutes at room temperature

  • Permeabilize with 0.5% Triton X-100 in PBS for 10 minutes

• Blocking and antibody incubation:

  • Block with 5% normal serum in PBS for 1 hour at room temperature

  • Incubate with primary anti-HOXB5 antibody at manufacturer-recommended dilution (typically 1:100 to 1:500) overnight at 4°C

  • Wash 3× with PBS

  • Incubate with fluorophore-conjugated secondary antibody for 1-2 hours at room temperature

  • Counterstain nuclei with DAPI

• Imaging considerations:

  • As a transcription factor, HOXB5 should primarily localize to the nucleus

  • Use confocal microscopy for optimal spatial resolution

  • Include controls stained with secondary antibody only

How can I use HOXB5/hoxb5a antibodies to study developmental processes in zebrafish?

For studies of zebrafish development using hoxb5a/b antibodies:

• Temporal expression analysis:

  • Perform immunostaining at different developmental stages (e.g., 14 hpf, 24 hpf, 30 hpf) to track dynamic expression patterns

  • Correlate with in situ hybridization data to compare protein and mRNA localization

• Combinatorial staining with tissue markers:

  • Co-stain with markers for specific tissues (e.g., Islet1 for endocrine pancreas, Myosin for somites)

  • Use transgenic reporter lines (e.g., Tg(isl1:mRFP)) for multi-color imaging

• Functional studies:

  • Combine antibody staining with morpholino knockdown or CRISPR/Cas9 gene editing approaches

  • Analyze phenotypes in structures known to express hoxb5a/b, such as foregut endoderm, lateral plate mesoderm (LPM), and neurons

How can I use HOXB5 antibodies to identify and isolate long-term hematopoietic stem cells (LT-HSCs)?

Based on findings that Hoxb5 marks LT-HSCs :

• Flow cytometry protocol:

  • Isolate bone marrow cells from appropriate tissue

  • Perform surface staining with HSC markers (Lin−, cKit+, Sca1+, CD150+, CD48−)

  • Fix and permeabilize cells for intracellular staining with anti-HOXB5 antibody

  • Use appropriate fluorophore-conjugated secondary antibody

  • Gate on HOXB5+ cells within the phenotypic HSC (pHSC) population

• Data analysis considerations:

  • Create proper gating strategy using FMO controls

  • Consider dividing HOXB5+ cells into Hoxb5hi and Hoxb5lo populations as they may have different functional properties

  • Validate by functional transplantation assays

• Imaging applications:

  • For tissue localization, combine with clearing methods like CUBIC to preserve fluorescence intensity

  • Quantify the number of HOXB5+ cells in bone marrow sections

  • Co-stain with niche markers like VE-cadherin to analyze microenvironmental interactions

What are common issues when using HOXB5/hoxb5a antibodies and how can they be addressed?

How do I interpret differences in HOXB5/hoxb5a expression patterns between different techniques?

When analyzing discrepancies:

• Between mRNA (in situ) and protein (antibody) detection:

  • Consider temporal delay between transcription and translation

  • Protein may persist longer than mRNA

  • Different sensitivities of detection methods may explain differences

  • Example: GFP-tagged hoxb5a protein may be detected in cells where mRNA is no longer detectable due to protein perdurance

• Between techniques (Western blot vs. immunofluorescence vs. flow cytometry):

  • Western blot reveals total protein content but loses spatial information

  • Immunofluorescence provides spatial context but may have lower sensitivity

  • Flow cytometry offers quantitative single-cell resolution but loses tissue context

  • Integrate data from multiple techniques for comprehensive understanding

• Between developmental stages or experimental conditions:

  • Document exact timing and conditions

  • Consider dynamic expression changes (e.g., hoxb5b expression shifts from LPM to endoderm during development)

  • Analyze both cell-autonomous and non-cell-autonomous effects

How are HOXB5/hoxb5a antibodies being used in chromatin studies?

Recent studies have revealed:

• Chromatin accessibility regulation:

  • Hoxa5 (paralog of Hoxb5) controls chromatin accessibility in mouse spinal cervical motor neuron subtypes

  • Differential analysis of ATAC-seq data reveals thousands of peaks that gain or lose accessibility upon Hoxa5 deletion

  • Antibodies can be used for ChIP-seq to correlate HOXB5 binding with changes in chromatin accessibility

• Transcription factor complex formation:

  • HOXB5 proteins interact with different co-factors (e.g., Pbx1, Scip) to regulate distinct gene sets

  • Co-immunoprecipitation with HOXB5 antibodies can identify protein complexes

  • Analysis of motif enrichment in HOXB5-bound regions can predict co-factor interactions

• Pioneer factor activity:

  • Some HOX proteins exhibit pioneer factor activity, opening previously inaccessible chromatin

  • Studying HOXB5's potential pioneer activity requires sequential ChIP-seq and ATAC-seq experiments

What are emerging approaches for developing more specific HOXB5/hoxb5a antibodies?

Based on recent advances in antibody technology :

• Computational design approaches:

  • Biophysics-informed modeling can predict antibody sequences with customized specificity profiles

  • Machine learning algorithms can identify optimal binding interfaces

• High-throughput screening methods:

  • Phage display combined with next-generation sequencing to identify highly specific binders

  • Golden Gate-based dual-expression vector systems for rapid screening of recombinant monoclonal antibodies

• Epitope mapping for increased specificity:

  • Targeting unique regions that distinguish between HOXB5 paralogs

  • Using structural information to design antibodies against conformation-specific epitopes

  • Developing antibodies against post-translationally modified forms of HOXB5