SALL2 Antibody

What is SALL2 and why is it significant for research?

SALL2 is a transcription factor member of the Spalt-like (SALL) family conserved across many organisms from nematodes to humans. The protein contains an N-terminal zinc finger domain of the C2HC type, a glutamine-rich region, and several double and triple zinc fingers of the C2H2 type throughout its structure . SALL2 plays crucial roles in:

• Neural development and differentiation

• Eye development before, during, and after optic fissure closure

• Regulation of cell migration processes

• Cell cycle progression and growth arrest

• Tumor suppression in certain contexts

SALL2 is particularly significant because it links neurotrophin receptor signaling with transcriptional events that regulate neuronal cell growth and development . Its dysregulation has been implicated in various disorders affecting the eyes, kidneys, and brain, as well as in several cancer types.

What isoforms of SALL2 exist and how do they differ functionally?

SALL2 exists in multiple isoforms, with the main ones being:

The E1 isoform contains a nuclear localization sequence and a conserved repressor motif that is absent in E1A, suggesting isoform-specific functions . These isoforms differ in the first 25 amino acids at the N-terminal domain and show distinct tissue expression patterns.

What applications are SALL2 antibodies typically used for?

SALL2 antibodies are utilized in multiple experimental techniques:

• Western Blotting: To detect SALL2 protein expression (observed molecular weight: 105 kDa)

• Immunoprecipitation: To study protein-protein interactions (e.g., SALL2-p75NTR complex)

• Chromatin Immunoprecipitation (ChIP): To investigate DNA-binding and transcriptional regulation

• Immunofluorescence: To study cellular localization and expression patterns

• ELISA: For quantitative detection of SALL2 protein

Which species reactivity should be considered when selecting SALL2 antibodies?

When selecting SALL2 antibodies, consider:

• Many commercial antibodies show reactivity with human, mouse, and rat SALL2

• Human SALL2 is homologous to mouse and rat SALL2 (90% identity)

• The domain of SALL2 that interacts with p75NTR is highly conserved among human, mouse, and rat (96% identity)

• SALL2 gene orthologs have been reported in mouse, rat, bovine, frog, and chimpanzee species

How can researchers validate the specificity of SALL2 antibodies?

To validate SALL2 antibody specificity, multiple approaches should be used:

• Peptide competition assays: Pretreatment with the peptide used to generate the antibody should eliminate the signal in Western blots and immunofluorescence .

• siRNA/CRISPR knockdown validation: Treatment of cells with SALL2-specific siRNA or using SALL2 knockout models should result in loss of the protein band identified as SALL2 on Western blots and reduced signal in immunofluorescence .

• Cross-species comparison: Analyze similar protein patterns in lysates from different species (e.g., mouse brain, human cell lines, rat PC12 cells) .

• Isoform-specific validation: For antibodies targeting specific isoforms, validation using isoform-specific knockout models (e.g., SALL2 E1A-knockout) is essential .

• Off-target analysis: Check predicted off-target sites when using CRISPR-generated knockout models for antibody validation .

What are the optimal conditions for SALL2 ChIP-Seq experiments?

Based on successful published protocols, optimal conditions for SALL2 ChIP-Seq include:

• Starting material:

  • Cell density: 1 × 10^6 cells per 100-mm dish

  • Chromatin amount: 25-40 μg per immunoprecipitation

• Sonication parameters:

  • Bioruptor plus sonicator (Diagenode): 40 cycles, 15s on/15s off, high potency

  • Alternative: Bioruptor Plus, 18 cycles, 15s on/20s off, 9W potency

  • Target DNA fragment size: 300-600 bp

• Antibody selection and amount:

  • 5 μg of Rabbit Polyclonal anti-SALL2 (Bethyl Cat# A303-208A)

  • Alternative: 2 μg of anti-FLAG for FLAG-tagged SALL2

• Controls:

  • Input controls: 1 μL for PCR reactions

  • Negative controls: SALL2 knockout cells for background assessment

  • Additional controls: 1 μg of H3 (anti-histone H3) and 1 μg of acH4 (anti-histone H4 acetylated)

• PCR validation: Target specific promoter regions of known SALL2 targets (e.g., ITGB1 promoter)

How do SALL2 protein-protein interactions impact experimental design?

SALL2 interaction with p75NTR and other proteins requires specific experimental considerations:

• Co-immunoprecipitation design:

  • SALL2 interacts with p75NTR through its death domain, requiring buffer conditions that maintain this interaction

  • The interaction can be demonstrated bidirectionally: immunoprecipitate SALL2 and blot for p75NTR, or immunoprecipitate p75NTR and blot for SALL2

• Stimulation conditions:

  • NGF treatment dissociates p75NTR/SALL2 complexes, so time-course experiments may be necessary to capture dynamic interactions

  • NGF also increases SALL2 expression through p75NTR, complicating interpretation of results

• Cellular compartment considerations:

  • After NGF stimulation, SALL2 translocates to the nucleus via TrkA-dependent mechanisms

  • Different cellular fractions (membrane, cytoplasmic, nuclear) should be analyzed separately

• Domain-specific interactions:

  • SALL2 interacts with p75NTR through multiple, non-contiguous amino acid sequences (amino acids 119-144 and 237-269)

  • This domain is not present in other SALL family members (SALL1, SALL3, SALL4)

What is the optimal protocol for detecting SALL2 in brain tissue?

For optimal detection of SALL2 in brain tissue:

• Tissue preparation:

  • Fresh brain tissue should be carefully dissected and immediately processed or flash-frozen

  • For fixed tissue, paraformaldehyde fixation followed by cryoprotection and sectioning is recommended

• Protein extraction:

  • Use lysis buffers containing protease inhibitors to prevent degradation

  • Brain tissue often requires stronger homogenization methods compared to cultured cells

• Western blotting:

  • Expected molecular weight: 105 kDa

  • Validate with positive controls (e.g., human HCT116 colon cancer cells or rat PC12 cells)

  • Include peptide competition controls to confirm specificity

• Immunofluorescence:

  • SALL2 colocalizes with p75NTR at the cell surface in mouse brain

  • Use confocal microscopy for improved resolution of subcellular localization

  • Include counterstains to identify specific brain regions or cell types

How should researchers study SALL2's role in cell migration?

To investigate SALL2's role in cell migration:

• Experimental models:

  • Immortalized Mouse Embryonic Fibroblasts (iMEFs) from Sall2 knockout mice provide a valuable model system

  • Alternatively, use CRISPR/Cas9 to generate SALL2 knockout in relevant cell lines

• Migration assays:

  • Wound healing (scratch) assays to assess collective cell migration

  • Transwell migration assays to quantify individual cell migration

  • Time-lapse microscopy to monitor dynamic changes in cell morphology and movement

• Cellular mechanisms to investigate:

  • Membrane protrusions formation

  • Cell detachment processes

  • Focal adhesion maturation and disassembly

  • FAK autophosphorylation at Y397

• Integrin analysis:

  • Measure integrin β1 mRNA and protein levels

  • Assess integrin availability at the cell surface

  • Examine Sall2 binding to the ITGB1 promoter using ChIP

• Rescue experiments:

  • Reintroduce specific SALL2 isoforms to determine their differential effects on migration

How should researchers interpret discrepancies in SALL2 function across different studies?

SALL2 shows context-dependent functions that require careful interpretation:

What approaches can be used to analyze SALL2 genomic binding patterns?

For comprehensive analysis of SALL2 genomic binding:

• Integration of multiple datasets:

  • Combine ChIP-seq data from different cell types (e.g., glioblastoma MGG8TPC, HEK293)

  • Compare binding patterns between wild-type and isoform-specific knockout cells

  • Integrate with histone modification data (H3K27ac, H3K4me3, H3K27me3)

• Bioinformatic analysis strategies:

  • Identify core transcription factor networks that work with SALL2

  • Compare binding occupancy between different SALL2 isoforms

  • Analyze conservation of binding sites across species

• Functional validation of binding sites:

  • Confirm direct binding using ChIP-qPCR of specific promoter regions (e.g., ITGB1)

  • Use reporter assays to validate transcriptional activity at binding sites

  • Perform gene expression analysis following SALL2 manipulation to confirm functional relevance

• Consideration of isoform-specific effects:

  • The long E1 and E1A isoforms likely contribute more significantly to transcriptional control

  • The short_E1A isoform shows low binding occupancy and forms less connected gene networks

  • Cytoplasmic localization of some isoforms may indicate non-transcriptional functions

What are the common challenges when detecting SALL2 in different experimental systems?

Researchers commonly encounter these challenges:

• Multiple isoform detection:

  • Different antibodies may detect specific or multiple SALL2 isoforms

  • Western blots may show multiple bands representing different isoforms

  • Verify which isoforms your antibody detects using isoform-specific knockout controls

• Low expression levels:

  • SALL2 expression is tissue-dependent and may be low in some contexts

  • In embryonic stem cells, SALL2 expression depends on culture conditions (almost no expression in naïve ESCs cultured in 2i/LIF, but expressed in serum/LIF medium)

  • Consider enrichment steps before detection in low-expressing samples

• Nuclear translocation dynamics:

  • SALL2 can shuttle between cytoplasm and nucleus in response to stimuli like NGF

  • Time-course experiments may be necessary to capture these dynamics

  • Nuclear/cytoplasmic fractionation may help resolve localization issues

• Cross-reactivity concerns:

  • Confirm specificity for SALL2 versus other SALL family members (SALL1, SALL3, SALL4)

  • The p75NTR interaction domain provides a unique epitope for SALL2-specific antibodies

How can researchers optimize SALL2 ChIP experiments for different target genes?

For successful SALL2 ChIP targeting different genes:

• Primer design considerations:

  • Design primers for proximal promoter regions (e.g., ITGB1 promoter SALL2-specific proximal I and II regions: -251/-127 and -631/-297)

  • Include negative control regions not bound by SALL2 (e.g., ITGB1 promoter region -1986/-1878)

• Protocol optimization:

  • Adjust crosslinking time based on target region accessibility

  • Optimize sonication conditions for consistent chromatin fragmentation

  • Consider dual crosslinking (formaldehyde plus additional crosslinker) for improved capture of indirect interactions

• Data analysis approaches:

  • Calculate enrichment relative to input and IgG controls

  • Compare binding in different cell states (e.g., before/after NGF treatment)

  • Correlate binding with gene expression changes

• Validation strategies:

  • Perform ChIP-qPCR on candidate binding sites identified by ChIP-seq

  • Use reporter gene assays to confirm functional relevance of binding sites

  • Compare binding in wild-type versus SALL2 isoform-specific knockout cells