BARHL2 Antibody

What is BARHL2 and what cellular functions does it regulate?

BARHL2 (BarH-Like Homeobox 2) is a 387 amino acid transcription factor that belongs to the BAR homeobox family of proteins with a calculated molecular weight of 42 kDa (observed molecular weight of 42-45 kDa in laboratory settings) . The protein contains a homeobox DNA-binding domain and localizes primarily in the nucleus where it functions as a transcriptional regulator . BARHL2 plays significant roles in several fundamental cellular processes including cell fate specification, differentiation, migration, and survival . Research indicates that BARHL2 may act as a transcription factor that binds to specific DNA consensus sequences to regulate gene expression during embryonic development . The protein's highly conserved sequence across multiple species (including human, chimpanzee, gorilla, monkey, rat, and others) suggests its evolutionarily important function in developmental and cellular processes .

What are the typical applications for BARHL2 antibodies in molecular research?

BARHL2 antibodies are employed in multiple experimental applications with specific methodological considerations for each technique:

For optimal results across these applications, researchers should validate antibody specificity in their specific experimental system and target tissues . Commercially available antibodies have been validated in specific cell lines including HepG2 and NIH/3T3 cells for Western blotting and immunofluorescence applications .

How should researchers select between polyclonal and monoclonal BARHL2 antibodies?

The selection between polyclonal and monoclonal BARHL2 antibodies should be based on specific experimental requirements:

Polyclonal antibodies (such as ABIN6735794 and 23976-1-AP) offer several advantages for BARHL2 detection:

• Recognition of multiple epitopes, providing stronger signal in applications like IHC

• Better tolerance to minor protein denaturation or conformation changes

• Generally more robust across different sample preparation methods

• Particularly useful for detecting BARHL2 in fixed tissue sections

For experiments requiring higher specificity to particular epitopes or quantitative analyses where consistency is critical, monoclonal antibodies would be preferred, though specific monoclonal options were not detailed in the provided search results.

What are the recommended protocols for BARHL2 immunohistochemistry?

For optimal BARHL2 detection in tissue sections, the following methodology is recommended:

• Section preparation: Use 4 μm sections of paraffin-embedded tissues

• Deparaffinization: Follow standard protocols for removing paraffin

• Antigen retrieval: Two validated methods:

  • Primary method: Incubation in TE buffer (pH 9.0)

  • Alternative method: Citrate buffer (pH 6.0) in a heated water bath (97°C) for 40 minutes

• Blocking: Immerse sections in Tris-buffered saline/5% bovine serum albumin solution for 10 minutes to reduce non-specific binding

• Primary antibody incubation: Apply BARHL2 antibody at dilution of 1:20-1:200 for 60 minutes

• Detection system: For fluorescent detection, use Alexa Fluor 568 secondary antibodies (e.g., goat anti-mouse IgG at 1:700 dilution for 30 minutes)

• Counterstaining: DAPI is recommended for nuclear visualization

This protocol has been validated for detecting BARHL2 in multiple tissues, including human testis tissue as noted in the Proteintech validation data .

How can researchers investigate BARHL2 methylation patterns in cancer research?

BARHL2 methylation analysis has emerged as a promising approach for cancer detection, particularly in gastric cancer research. The methodology involves:

• Primer design: Target the CpG island encompassing the transcription start site of BARHL2 for bisulfite-pyrosequencing analysis

• DNA extraction: Obtain DNA from relevant samples (e.g., gastric wash, gastric juice-derived exosomal DNA)

• Methylation analysis: Perform bisulfite conversion followed by pyrosequencing to quantify methylation levels

• Data interpretation: Compare methylation levels between:

  • Cancer vs. normal tissues

  • Pre-treatment vs. post-treatment samples

  • Different stages of cancer progression

Research has demonstrated that high levels of BARHL2 methylation correlate with reduced BARHL2 expression in gastric cancer cell lines. Treatment with 5-aza-2′-deoxycytidine can restore BARHL2 expression in these methylated cell lines, suggesting epigenetic regulation of this gene . The BARHL2 methylation approach has shown promising results in gastric cancer detection, with one study reporting an area under the curve of 0.923 with 90% sensitivity and 100% specificity when distinguishing gastric cancer patients from non-cancer controls using gastric juice-derived exosomal DNA .

What experimental approaches can be used to study BARHL2 function in cell lines?

To investigate BARHL2 function in cellular processes, researchers can employ several established experimental approaches:

• Transfection with BARHL2 expression vectors:

  • Utilize Myc-DDK-tagged pCMV6-BARHL2 expression vectors

  • Electroporation techniques with Nucleofector II Device and appropriate kits have shown success

  • Confirm expression by Western blotting using anti-BARHL2 antibodies

• Colony formation assays:

  • Plate cells (0.5 × 10^5) in culture dishes 24 hours before transfection

  • After transfection, maintain cells in G418-containing medium (concentration dependent on cell line: 0.2 mg/ml for MKN7 and 0.6 mg/ml for MKN45)

  • Culture for 14 days before staining with Giemsa or crystal violet

  • Quantify colonies using NIH Image software or equivalent

• Flow cytometry analysis for cell cycle studies:

  • Synchronize cells through medium depletion for 3 days

  • Transfect with BARHL2 expression vector

  • After 48 hours, fix and stain cells using a Cell Cycle Phase Determination Kit

  • Analyze by flow cytometry to determine impact on cell cycle progression

• Gene expression analysis:

  • Perform RNA extraction following BARHL2 overexpression or knockdown

  • Conduct qRT-PCR or RNA-seq to identify genes regulated by BARHL2

  • Validate findings with ChIP assays to confirm direct binding targets

These experimental approaches have been successfully employed to characterize BARHL2's role in cellular proliferation and differentiation in the context of cancer research .

What are the technical considerations for Western blot detection of BARHL2?

Successful Western blot detection of BARHL2 requires optimization of several critical parameters:

• Sample preparation:

  • Effective lysis buffers containing protease inhibitors are essential

  • Nuclear extraction protocols may improve detection due to BARHL2's nuclear localization

  • Denaturation conditions should be optimized (typical molecular weight: 42-45 kDa)

• Antibody selection and dilution:

  • For polyclonal antibodies like 23976-1-AP: Use 1:200-1:1000 dilution

  • Positive control lysates: HepG2 cells, NIH/3T3 cells have confirmed BARHL2 expression

  • Species compatibility should be confirmed - human and mouse reactivity is well-established

• Detection optimization:

  • Secondary antibody selection should match host species (typically rabbit IgG)

  • Both standard ECL and fluorescent detection systems are compatible

  • Expected band size verification: BARHL2 typically appears at 42-45 kDa

• Troubleshooting considerations:

  • If detection is difficult, consider membrane stripping and re-probing with higher antibody concentration

  • Extended exposure times may be necessary for low abundance expression

  • Cross-reactivity validation is recommended when working with novel cell types

Positive Western blot results have been consistently obtained with HepG2 and NIH/3T3 cell lysates using the 23976-1-AP antibody at appropriate dilutions .

How can researchers validate BARHL2 antibody specificity?

Comprehensive validation of BARHL2 antibody specificity should employ multiple complementary approaches:

• Positive and negative control samples:

  • Positive controls: HepG2 cells, NIH/3T3 cells, human testis tissue

  • Negative controls: Tissues or cell lines with confirmed absence of BARHL2 expression

• Overexpression validation:

  • Transfect cells with Myc-DDK-tagged pCMV6-BARHL2 expression vectors

  • Compare antibody signal between transfected and non-transfected cells

  • Co-staining with anti-tag antibodies to confirm specificity

• Knockdown validation:

  • Perform siRNA or shRNA knockdown of BARHL2

  • Verify signal reduction via Western blot or immunostaining

• Multiple detection methods:

  • Compare results across different applications (WB, IHC, IF)

  • Concordance across methods strengthens validation

  • Peptide competition assays can confirm epitope specificity

• Cross-species reactivity assessment:

  • Test antibody against BARHL2 from multiple species

  • Compare observed reactivity with predicted reactivity based on sequence homology

  • Known cross-reactivity: Human (100%), Guinea pig (100%), Mouse (92%), Rabbit (92%), Pig (92%), Dog (85%), Bovine (85%)

These validation steps ensure experimental reliability and reproducibility when studying BARHL2 across different experimental systems and species.

What is the relationship between BARHL2 expression and cancer pathogenesis?

Research suggests BARHL2 may have significant roles in cancer development and progression:

• Epigenetic regulation in gastric cancer:

  • High levels of BARHL2 methylation are detected in gastric cancer cell lines, correlating with low BARHL2 expression

  • BARHL2 methylation is elevated in early gastric cancer patients and decreases after endoscopic resection

  • BARHL2 methylation appears independent of Helicobacter pylori infection, suggesting it as a potential H. pylori-independent biomarker

• Functional implications:

  • As a homeodomain transcription factor, BARHL2 regulates gene expression affecting cell fate specification, differentiation, migration, and survival

  • Normal gastric epithelial cells show BARHL2 nuclear immunoreactivity

  • Loss of this expression through methylation may contribute to cellular transformation

• Diagnostic potential:

  • BARHL2 methylation analysis using gastric juice-derived exosomal DNA has demonstrated high sensitivity (90%) and specificity (100%) for gastric cancer detection

  • This suggests potential applications as a non-invasive biomarker

• Experimental approaches for further investigation:

  • Colony formation assays following BARHL2 restoration in cancer cell lines

  • Cell cycle analysis through flow cytometry after BARHL2 expression

  • Gene expression profiling to identify downstream targets

Further research is needed to fully elucidate BARHL2's role in other cancer types and its potential as a therapeutic target or diagnostic marker.

How can researchers optimize immunofluorescence detection of BARHL2?

For optimal immunofluorescence detection of BARHL2 in cells and tissues, consider the following protocol optimizations:

• Sample preparation:

  • For tissues: Use 4 μm paraffin sections with appropriate antigen retrieval

  • For cells: Fix with 4% paraformaldehyde and permeabilize with 0.1-0.5% Triton X-100

• Antibody parameters:

  • Primary antibody dilution: 1:200-1:800 for most BARHL2 antibodies

  • Incubation conditions: 60 minutes at room temperature or overnight at 4°C

  • Secondary antibody selection: Alexa Fluor 568 goat anti-mouse/rabbit IgG at 1:700 dilution

• Detection optimization:

  • Expected pattern: Nuclear localization consistent with BARHL2's function as a transcription factor

  • Counterstaining: DAPI for nuclear visualization

  • Mounting media: Use anti-fade reagents to preserve fluorescence signal

• Controls and validation:

  • Positive controls: HepG2 cells show consistent BARHL2 expression

  • Negative controls: Secondary antibody only

  • Competition controls: Pre-incubation with immunizing peptide

• Troubleshooting weak signals:

  • Increase primary antibody concentration

  • Extend incubation time

  • Optimize antigen retrieval conditions (test both TE buffer pH 9.0 and citrate buffer pH 6.0)

  • Consider signal amplification systems for low-abundance targets

These optimizations have been validated for detecting BARHL2 in both cell lines and tissue specimens, enabling reliable visualization of this transcription factor in its native nuclear compartment.

What are the best approaches for studying BARHL2 in different species?

When studying BARHL2 across different species, researchers should consider both sequence homology and technical adaptations:

• Species reactivity considerations:

  • Highest sequence homology (100%): Human, Chimpanzee, Gorilla, Monkey, Galago, Marmoset, Rat, Panda, Horse, Guinea pig

  • High homology (92%): Gibbon, Mouse, Rabbit, Pig

  • Moderate homology (84-86%): Elephant, Dog, Bovine, Bat

• Antibody selection strategy:

  • For cross-species applications, select antibodies targeting highly conserved regions

  • The aa165-214 region of BARHL2 shows excellent conservation across mammalian species

  • Validate antibody in each new species before conducting full experiments

• Application-specific recommendations:

  • Western blotting: May require species-specific loading control antibodies

  • IHC/IF: Optimize antigen retrieval conditions for each species' tissue fixation protocols

  • Consider species-specific secondary antibodies to minimize background

• Controls for cross-species work:

  • Include tissues/cells with known BARHL2 expression from well-characterized species

  • Consider parallel experiments with species-specific antibodies when available

  • Sequence alignment analysis to predict cross-reactivity before experimental work

This systematic approach enables reliable BARHL2 detection across multiple species, facilitating comparative studies of this evolutionarily conserved transcription factor.

How should researchers interpret contradictory results in BARHL2 studies?

When confronted with contradictory results in BARHL2 research, consider these analytical approaches:

• Technical factors assessment:

  • Antibody variables: Different epitope targets between antibodies

  • Sample preparation differences: Fixation, antigen retrieval, and extraction methods

  • Detection system sensitivity variations

• Biological context considerations:

  • Cell type/tissue specificity: BARHL2 function may vary between tissues

  • Developmental stage: Expression patterns may change during development

  • Pathological state: Disease conditions may alter expression or localization

• Methodological reconciliation strategy:

  • Multi-method validation: Compare results across different techniques (WB, IHC, qPCR)

  • Quantitative analysis: Use appropriate statistical methods to determine significance

  • Independent repetition: Confirm findings with biological and technical replicates

• Common contradictions and resolutions:

  • Expression level discrepancies: Often resolved through quantitative normalization

  • Subcellular localization differences: May reflect genuine biological variation or fixation artifacts

  • Functional impact variations: May indicate context-dependent roles requiring specific co-factors

By systematically analyzing the source of contradictions, researchers can develop more robust experimental designs that account for the complexity of BARHL2 biology across different experimental systems.

What emerging technologies can enhance BARHL2 research?

Several cutting-edge technologies offer new opportunities for advancing BARHL2 research:

• Single-cell analysis approaches:

  • Single-cell RNA-seq for cell-specific BARHL2 expression profiling

  • Single-cell ATAC-seq to study chromatin accessibility at BARHL2 loci

  • Mass cytometry for multi-parameter protein expression analysis

• CRISPR-based technologies:

  • CRISPR-Cas9 for precise BARHL2 gene editing

  • CRISPRi/CRISPRa for reversible modulation of BARHL2 expression

  • CRISPR screens to identify genes in BARHL2 regulatory networks

• Advanced imaging technologies:

  • Super-resolution microscopy for detailed subcellular localization

  • Live-cell imaging with fluorescently tagged BARHL2

  • Spatial transcriptomics for tissue-context expression analysis

• Computational and systems biology:

  • Network analysis to position BARHL2 in developmental pathways

  • Machine learning approaches to predict BARHL2 target genes

  • Structural modeling of BARHL2-DNA interactions

These emerging technologies will enable more precise characterization of BARHL2's function in normal development and disease contexts, potentially revealing new therapeutic targets and diagnostic approaches.

How might BARHL2 research contribute to clinical applications?

BARHL2 research has several promising translational applications:

• Cancer diagnostics:

  • BARHL2 methylation analysis in liquid biopsies (e.g., gastric wash or gastric juice-derived exosomal DNA)

  • Development of antibody-based diagnostic panels incorporating BARHL2

  • Early detection systems for gastric cancer based on BARHL2 methylation patterns

• Therapeutic target development:

  • Epigenetic modulation to restore BARHL2 expression in cancers

  • Small molecule screening to identify compounds affecting BARHL2 function

  • Development of gene therapy approaches targeting BARHL2 pathways

• Prognostic biomarker applications:

  • Correlation of BARHL2 expression/methylation with disease progression

  • Integration into multi-marker prognostic panels

  • Monitoring treatment response through BARHL2 methylation changes

• Developmental disorder insights:

  • Understanding BARHL2's role in cell fate determination may provide insights into developmental disorders

  • Potential applications in regenerative medicine based on BARHL2's role in cellular differentiation