SOHLH2 Antibody

What is SOHLH2 and what are its primary functions in normal tissues?

SOHLH2 is a transcription factor with a molecular mass of approximately 32 kDa that belongs to the basic-loop-helix (bHLH) protein transcription factor family. It functions as a transcriptional regulator by binding to conserved E-box sequences in target gene promoters .

In normal physiology, SOHLH2 plays several critical roles:

• Regulates both male and female germline differentiation

• Suppresses genes involved in spermatogonial stem cell maintenance

• Induces genes essential for spermatogonial differentiation

• Coordinates oocyte differentiation without affecting meiosis I

• Essential for synaptonemal complex formation during meiosis

• Highly expressed in various human adult normal tissues

The importance of SOHLH2 in reproduction is highlighted by studies showing that SOHLH2 knockout mice exhibit infertility due to disrupted germ cell development, with specific defects in synaptonemal complex formation and meiotic progression .

What applications are validated for SOHLH2 antibodies in research?

SOHLH2 antibodies have been validated for multiple experimental applications:

Rabbit polyclonal SOHLH2 antibodies have been successfully used with human samples in multiple publications, with immunogens corresponding to recombinant fragment proteins within human SOHLH2 amino acids 1-200 .

How should researchers interpret SOHLH2 expression patterns in different tissues?

When analyzing SOHLH2 expression using antibody-based detection methods, researchers should consider:

• Normal expression profile: SOHLH2 is primarily expressed in germ cells but also detected in various adult tissues

• Subcellular localization: Predominantly nuclear localization reflecting its function as a transcription factor

• Expression in cancer tissues: Generally downregulated in various cancers including renal cell carcinoma, ovarian cancer, and breast cancer

• Correlation with clinical parameters: SOHLH2 expression negatively correlates with tumor grade, tumor size, and metastasis

For rigorous interpretation, researchers should:

• Include proper positive controls (testis/ovary tissue)

• Compare expression across multiple samples

• Correlate with other relevant markers

• Consider quantitative analysis methods (H-score, digital image analysis)

• Validate findings with orthogonal techniques (qPCR, Western blotting)

What controls should be included when working with SOHLH2 antibodies?

Proper experimental controls are essential for reliable results when using SOHLH2 antibodies:

Essential controls for all applications:

• Positive tissue control: Testis or ovary tissues known to express SOHLH2

• Negative control: Tissues where SOHLH2 is not expressed or SOHLH2 knockout tissues

• Antibody isotype control: To assess non-specific binding

• Primary antibody omission: To evaluate background staining

Application-specific controls:

• For Western blotting:

  • SOHLH2 overexpression lysates

  • SOHLH2 knockdown/knockout lysates

  • Loading control (GAPDH, Actin)

• For IHC/IF:

  • Peptide competition assay

  • Serial dilutions of primary antibody

  • Secondary antibody only control

• For ChIP assays:

  • Input control

  • IgG control

  • Positive control regions (known SOHLH2 targets like SYCP1)

  • Negative control regions (non-E-box containing regions)

How can SOHLH2 antibodies be validated for specificity?

Thorough validation of SOHLH2 antibody specificity is crucial for generating reliable research data:

Molecular weight verification:

• SOHLH2 should appear as a band at approximately 32 kDa on Western blots

• Check for absence of non-specific bands

Genetic validation approaches:

• Compare staining between wild-type and SOHLH2 knockout tissues

• Assess signal in SOHLH2 overexpression vs. knockdown cell models

• Perform siRNA-mediated knockdown and verify reduced signal

Epitope-specific validation:

• Conduct peptide competition assays using the immunizing peptide

• Compare results from antibodies targeting different SOHLH2 epitopes

• Verify recognition of recombinant SOHLH2 protein

Functional validation:

• Confirm nuclear localization consistent with transcription factor function

• Verify expected expression pattern in germline tissues

• Demonstrate expected changes in signal with biological manipulations

How can SOHLH2 antibodies be utilized to investigate its tumor suppressor function?

SOHLH2 functions as a tumor suppressor in various cancers. Researchers can employ SOHLH2 antibodies to investigate this role through these methodological approaches:

Expression correlation studies:

• Perform IHC on tumor microarrays to correlate SOHLH2 expression with:

  • Tumor grade and stage

  • Proliferation markers (Ki67)

  • Patient survival outcomes

  • Microvessel density (as SOHLH2 negatively correlates with MVD)

Functional studies:

• Generate SOHLH2-overexpressing and knockdown cell models

• Confirm expression changes by Western blotting

• Assess effects on:

  • Cell proliferation (growth curves, colony formation)

  • Migration (wound healing assays)

  • Invasion (transwell assays)

  • EMT markers (E-cadherin, N-cadherin, ZO-1)

Mechanistic investigations:

• Use ChIP with SOHLH2 antibodies to identify direct target genes

• Perform co-immunoprecipitation to identify protein interaction partners

• Analyze downstream effectors:

  • HIF1α pathway components (for angiogenesis regulation)

  • DNMT3a and Klotho (for epigenetic regulation in RCC)

In vivo validation:

• Establish xenograft models with SOHLH2-modulated cancer cells

• Analyze tumors for growth rate, volume, and weight

• Assess metastatic potential to lungs and liver

• Perform IHC to verify maintained SOHLH2 expression and evaluate effects on proliferation and EMT markers

What methods should be employed to study SOHLH2's role in meiosis?

SOHLH2 is essential for synaptonemal complex formation during meiosis. Researchers can investigate this function using the following approaches:

Developmental expression analysis:

• Collect testicular samples at different timepoints (7-21 days postpartum)

• Perform Western blotting to determine SOHLH2 expression dynamics

• Compare with expression of known meiotic markers (SYCP1, SYCP3, SPO11)

Structural studies:

• Perform co-immunofluorescence for:

  • SOHLH2 and SYCP1 (transverse element of synaptonemal complex)

  • SOHLH2 and SYCP3 (lateral element of synaptonemal complex)

  • SOHLH2 and γH2AX (marker of meiotic DNA double-strand breaks)

• Use electron microscopy to analyze synaptonemal complex ultrastructure

Molecular regulation studies:

• Conduct ChIP assays with SOHLH2 antibodies to identify binding to meiotic gene promoters

• Perform luciferase reporter assays to evaluate SOHLH2's effect on target gene transcription

• Use EMSA to confirm direct binding to E-box sequences in promoters:

  • Prepare biotin-labeled oligonucleotides containing E-box motifs

  • Use in vitro translated SOHLH2 for binding reactions

  • Confirm specificity with competition assays

Genetic model analysis:

• Compare synaptonemal complex formation between wild-type and SOHLH2 knockout mice

• Analyze changes in expression of synaptonemal complex components (SYCP1, SYCP3)

• Assess meiotic progression defects using stage-specific markers

How can researchers investigate SOHLH2's role in regulating epithelial-mesenchymal transition?

SOHLH2 inhibits epithelial-mesenchymal transition (EMT) in cancer cells. To study this function:

Expression analysis in cancer samples:

• Perform multiplex immunofluorescence for SOHLH2 and EMT markers:

  • Epithelial markers: E-cadherin, ZO-1, Claudin

  • Mesenchymal markers: N-cadherin, Vimentin, Fibronectin, ZEB-1

• Focus analysis on tumor invasive front

• Quantify marker expression using digital image analysis

In vitro modulation studies:

• Generate SOHLH2-overexpressing and knockdown cell models

• Validate expression by Western blotting and qPCR

• Assess EMT marker expression changes using:

  • Western blotting (protein levels)

  • qPCR (mRNA expression)

  • Immunofluorescence (subcellular localization)

• Document morphological changes via phase contrast microscopy

Functional assessments:

• Wound healing assays to measure migration capacity

• Transwell invasion assays to quantify invasive potential

• 3D culture systems to evaluate morphological changes

• Time-lapse imaging to capture dynamic phenotypic transitions

Mechanistic studies:

• ChIP-seq to identify direct EMT-related target genes

• Pathway analysis focusing on known EMT regulators

• Rescue experiments combining SOHLH2 modulation with EMT inducer treatment (e.g., TGF-β)

What approaches are optimal for studying SOHLH2's role in angiogenesis inhibition?

SOHLH2 suppresses angiogenesis by downregulating HIF1α. To investigate this function:

Clinical correlation studies:

• Perform IHC on tumor tissue arrays for:

  • SOHLH2 expression

  • Microvessel density (using CD31/CD34)

  • HIF1α expression

• Analyze correlations between these parameters and clinical outcomes

• Quantify using digital image analysis for objective assessment

Molecular mechanism studies:

• ChIP assays to determine if SOHLH2 directly binds to the HIF1α promoter:

  • Design primers flanking E-box motifs in the HIF1α promoter

  • Perform ChIP-qPCR to quantify enrichment

• Luciferase reporter assays with HIF1α promoter constructs:

  • Test wild-type and E-box mutant promoters

  • Evaluate dose-dependent effects of SOHLH2

• Analyze expression of HIF1α-regulated pro-angiogenic genes:

  • qPCR for mRNA expression

  • Western blotting for protein levels

  • ELISA for secreted factors

Functional angiogenesis assays:

• Generate conditioned media from SOHLH2-modulated cancer cells

• Use conditioned media in endothelial cell assays:

  • Tube formation assays

  • Migration assays

  • Proliferation assays

• Quantify endothelial cell behavior using appropriate metrics:

  • Total tube length

  • Number of branch points

  • Migration distance

  • Proliferation rate

In vivo angiogenesis models:

• Establish xenograft models with SOHLH2-modulated cancer cells

• Analyze tumor vasculature by IHC for CD31

• Perform Matrigel plug assays

• Consider intravital microscopy for dynamic vessel formation assessment

How can researchers identify and validate SOHLH2 transcriptional targets?

As a transcription factor, SOHLH2 regulates gene expression by binding to E-box motifs. To identify its targets:

Genome-wide binding site identification:

• Perform ChIP-seq with validated SOHLH2 antibodies:

  • Optimize crosslinking conditions (1% formaldehyde, 10 minutes)

  • Sonicate chromatin to 200-500 bp fragments

  • Immunoprecipitate with SOHLH2 antibody

  • Include appropriate controls (input, IgG)

• Analyze data for enriched motifs:

  • Focus on canonical E-box motifs (CANNTG)

  • Identify genomic distribution of binding sites

  • Annotate peaks to nearest genes

Target validation approaches:

• ChIP-qPCR for candidate target genes:

  • Design primers flanking predicted binding sites

  • Calculate percent input or fold enrichment over IgG

  • Compare binding in different cell types/conditions

• Luciferase reporter assays:

  • Clone promoter regions containing SOHLH2 binding sites

  • Test wild-type and mutated E-box sequences

  • Evaluate dose-dependent effects of SOHLH2

• EMSA to confirm direct binding:

  • Use in vitro translated SOHLH2

  • Prepare biotin-labeled probes containing E-box motifs

  • Include competition assays and supershift with SOHLH2 antibody

Functional validation:

• Modulate SOHLH2 expression and assess target gene changes:

  • qPCR for mRNA expression

  • Western blotting for protein levels

• Rescue experiments:

  • Simultaneously modulate SOHLH2 and target genes

  • Assess phenotypic outcomes

Research has demonstrated that SOHLH2 directly regulates SYCP1 expression by binding to E-box sequences in its promoter .

What strategies can be employed to investigate SOHLH2's epigenetic regulatory functions?

SOHLH2 has been shown to regulate gene expression through epigenetic mechanisms, particularly in renal cell carcinoma:

DNA methylation studies:

• Investigate SOHLH2's effect on DNA methyltransferases:

  • Perform Western blot and qPCR for DNMT3a in SOHLH2-modulated cells

  • Co-immunoprecipitation to detect potential protein interactions

  • ChIP to determine if SOHLH2 directly regulates DNMT3a expression

• Analyze methylation status of target genes:

  • Bisulfite sequencing of candidate target promoters (e.g., Klotho)

  • Methylation-specific PCR

  • Genome-wide methylation profiling (reduced representation bisulfite sequencing or methylation arrays)

Correlation studies:

• Perform multiplex IHC to assess correlations between:

  • SOHLH2 expression

  • DNMT3a expression

  • Klotho expression

  • Methylation markers (5-methylcytosine)

• Quantify using digital image analysis

• Correlate with clinical parameters

Functional validation:

• Rescue experiments:

  • Combine SOHLH2 overexpression with DNMT3a modulation

  • Assess effects on target gene expression and methylation

  • Evaluate phenotypic outcomes (proliferation, migration, etc.)

• Demethylating agent studies:

  • Treat SOHLH2-deficient cells with 5-aza-2'-deoxycytidine

  • Assess restoration of target gene expression

  • Compare with SOHLH2 re-expression effects

Mechanistic investigations:

• ChIP-seq for histone modifications in SOHLH2-modulated cells

• RNA-seq to identify global expression changes

• Integrate methylation, histone modification, and expression data to build comprehensive regulatory networks

How can SOHLH2 antibodies be used in multiplexed imaging approaches?

Multiplexed imaging allows simultaneous visualization of multiple proteins. For SOHLH2 studies:

Panel design considerations:

• Select markers relevant to SOHLH2 function:

  • EMT markers (E-cadherin, N-cadherin)

  • Proliferation markers (Ki67)

  • Angiogenesis markers (CD31, HIF1α)

  • Epigenetic regulators (DNMT3a)

  • Target genes (Klotho, SYCP1)

• Address technical considerations:

  • Use antibodies from different host species when possible

  • For same-species antibodies, employ sequential staining with complete stripping

  • Balance signal strengths across markers

  • Consider tyramide signal amplification for low-abundance targets

Optimization steps:

• Validate each antibody individually:

  • Determine optimal concentration

  • Establish appropriate antigen retrieval method

  • Confirm specificity with appropriate controls

• Test antibody combinations for compatibility

• Optimize signal separation and detection parameters

Controls for multiplexed imaging:

• Single-color controls for determining spectral overlap

• Fluorescence-minus-one (FMO) controls

• Serial sections stained with individual antibodies

Analysis approaches:

• Cell segmentation for quantitative single-cell analysis

• Colocalization analysis to quantify spatial relationships

• Neighborhood analysis to assess cellular interactions

• Spatial statistics to identify significant patterns

What troubleshooting strategies can address common challenges with SOHLH2 antibodies?

When working with SOHLH2 antibodies, researchers may encounter several challenges:

Low signal intensity:

• Optimize antigen retrieval:

  • Test different buffers (citrate pH 6.0, EDTA pH 9.0)

  • Adjust retrieval duration and temperature

• Signal amplification approaches:

  • Tyramide signal amplification

  • Biotin-streptavidin systems

  • Higher antibody concentration

• Extended primary antibody incubation (overnight at 4°C)

• Use fresh tissue samples or properly stored specimens

High background:

• Optimize blocking:

  • Increase blocking time (1-2 hours)

  • Test different blocking agents (BSA, normal serum, commercial blockers)

  • Add detergent to reduce non-specific binding

• Antibody optimization:

  • Titrate to determine optimal concentration

  • Reduce incubation time or temperature

  • Pre-absorb antibody with non-specific proteins

• Increase washing stringency:

  • Additional wash steps

  • Extended wash times

  • Higher detergent concentration

Inconsistent results:

• Standardize sample processing:

  • Consistent fixation times

  • Uniform tissue thickness

  • Standardized antigen retrieval

• Include positive and negative controls in each experiment

• Prepare fresh reagents for each experiment

• Use validated antibody lots with consistent performance

• Consider automated staining platforms for reproducibility