SSH3 Antibody

What is SSH3 and why is it important in cellular research?

SSH3 (Slingshot homolog 3) is a 659 amino acid protein that functions as a protein phosphatase and localizes to both the nucleus and cytoplasm. It is a human homolog of the Drosophila slingshot (ssh) protein . SSH3 plays a critical role in regulating actin filament dynamics by controlling proteins such as actin-depolymerizing factor (ADF) and Cofilin. The ADF/Cofilin family consists of stimulus-responsive mediators that rapidly depolymerize and disassemble F-actin, and SSH3 catalytically dephosphorylates these proteins to reactivate them . This regulation of actin dynamics is essential for numerous cellular processes including cell migration, morphogenesis, and cytokinesis, making SSH3 an important target for cytoskeletal research.

What are the structural and functional characteristics of SSH3?

SSH3 contains one tyrosine-protein phosphatase domain and is expressed as five isoforms due to alternative splicing events . The protein has a calculated molecular weight of approximately 73 kDa, though it typically appears at 90-95 kDa in Western blots due to post-translational modifications . SSH3 is encoded by a gene located on chromosome 11q13.2 (Gene ID: 54961) . Functionally, SSH3 acts as a phosphatase that regulates the phosphorylation state of ADF/Cofilin proteins, which are critical for actin filament turnover and reorganization. This regulation affects cellular processes dependent on cytoskeletal dynamics including cell division, motility, and morphological changes.

What types of SSH3 antibodies are available for research applications?

Researchers have access to several types of SSH3 antibodies:

• Mouse monoclonal antibodies: Highly specific antibodies raised against human SSH3, including IgG2a kappa isotype options

• Rabbit polyclonal antibodies: Offer broader epitope recognition, useful for various applications and detection of multiple isoforms

• Conjugated antibodies: Available with various conjugations including agarose, HRP, PE, FITC, and multiple Alexa Fluor® conjugates for specialized applications

Each antibody type offers different advantages depending on the experimental requirements, with monoclonals providing high specificity and polyclonals offering robust detection across applications.

Which applications are SSH3 antibodies validated for?

SSH3 antibodies have been validated for numerous research applications:

Each application requires specific optimization of antibody concentration, incubation conditions, and detection methods to achieve optimal results .

How should I optimize Western blot protocols for SSH3 detection?

For optimal Western blot detection of SSH3:

• Sample preparation: Use appropriate lysis buffers containing phosphatase inhibitors to preserve SSH3's phosphorylation state

• Protein loading: Load 20-50 μg of total protein per lane

• Gel percentage: Use 8-10% SDS-PAGE gels to properly resolve the 90-95 kDa SSH3 protein

• Transfer conditions: Transfer to PVDF membrane at constant voltage (100V) for 90-120 minutes in cold transfer buffer

• Blocking: Block with 5% non-fat milk or BSA in TBST for 1 hour at room temperature

• Primary antibody: Dilute antibody 1:1000-1:4000 in blocking buffer and incubate overnight at 4°C

• Secondary antibody: Use appropriate HRP-conjugated secondary antibody at 1:5000-1:10000 dilution

• Detection: Both chemiluminescent and fluorescent detection methods are compatible with SSH3 antibodies

Note that SSH3 may appear at a higher molecular weight (90-95 kDa) than calculated (73 kDa) due to post-translational modifications .

Why might SSH3 appear at a different molecular weight than expected?

The observed molecular weight of SSH3 (90-95 kDa) often differs from the calculated weight (73 kDa) . This discrepancy can be attributed to:

• Post-translational modifications: Phosphorylation at multiple sites can significantly increase apparent molecular weight

• Glycosylation: N-linked or O-linked glycosylation can add substantial mass

• Alternative splicing: SSH3 is expressed as five different isoforms, which may run at different molecular weights

• Sample preparation: Incomplete denaturation or reduction can affect migration patterns

• Gel concentration: Too high or too low percentage gels may cause aberrant migration

When interpreting bands, researchers should confirm specificity using positive controls such as HeLa or HT-29 cell lysates, which have been validated for SSH3 expression . Western blot analysis using immunogen-specific blocking peptides can also help confirm band specificity.

What are common pitfalls in immunohistochemistry with SSH3 antibodies?

When performing IHC with SSH3 antibodies, researchers should be aware of these common challenges:

• Antigen retrieval optimization: SSH3 antibodies may require specific antigen retrieval methods for optimal staining

  • For rabbit polyclonal antibodies: Use TE buffer pH 9.0 or citrate buffer pH 6.0

  • For mouse monoclonal antibodies: Follow manufacturer-specific recommendations

• Specificity concerns:

  • Background staining can occur due to endogenous biotin or peroxidase activity

  • Include appropriate blocking steps (hydrogen peroxide treatment, avidin/biotin blocking)

  • Always include proper negative controls (isotype control or secondary-only control)

• Signal intensity issues:

  • Titrate antibody concentration (typically 1:50-1:500)

  • Optimize incubation time and temperature

  • Consider amplification systems for low-abundance targets

• Tissue-specific considerations:

  • SSH3 has been validated in salivary gland , colon cancer, and breast cancer tissues

  • Different fixation protocols may affect epitope availability

Methodological recommendation: Perform parallel validation using two different SSH3 antibodies (monoclonal and polyclonal) on serial sections to confirm staining patterns.

How can SSH3 antibodies be utilized in studying actin cytoskeleton dynamics?

SSH3 antibodies can be powerful tools for studying actin cytoskeleton regulation through several advanced approaches:

• Co-localization studies:

  • Use SSH3 antibodies in dual immunofluorescence with F-actin markers (phalloidin) and phospho-Cofilin antibodies

  • Analyze spatial relationships between SSH3, actin structures, and its substrates during cellular events such as migration or division

• Phosphatase activity assays:

  • Immunoprecipitate SSH3 using validated antibodies

  • Measure phosphatase activity against purified phospho-Cofilin substrates

  • Compare activity under different cellular conditions or treatments

• Live-cell imaging:

  • Validate SSH3 antibody specificity for use as a basis for generating fluorescent protein-tagged SSH3 constructs

  • Monitor dynamic interactions between SSH3 and cytoskeletal components during cellular processes

• Stimulus-response experiments:

  • Analyze SSH3 localization and activity following cytoskeletal-disrupting agents

  • Quantify changes in SSH3-dependent dephosphorylation of Cofilin in response to stimuli

These approaches allow researchers to dissect the temporal and spatial regulation of actin dynamics by SSH3 and its relationship to cellular function.

What considerations are important when studying SSH3 in disease models?

When investigating SSH3 in disease contexts, researchers should consider:

• Expression level variations:

  • Use validated antibodies for accurate quantification of SSH3 in different disease states

  • Perform both Western blot and IHC analyses to correlate expression levels with tissue localization

• Phosphorylation state analysis:

  • Since SSH3 is both regulated by phosphorylation and regulates phosphorylation of substrates, use phospho-specific antibodies in conjunction with total SSH3 antibodies

  • Consider phosphatase treatment controls to confirm phosphorylation-dependent signals

• Isoform-specific detection:

  • Be aware that SSH3's five isoforms may be differentially expressed in disease states

  • Use antibodies recognizing conserved regions or isoform-specific antibodies as appropriate

• Sample preparation preservation:

  • Phosphorylation states can be rapidly lost during sample processing

  • Use appropriate phosphatase inhibitors and rapid processing protocols

• Cross-reactivity considerations:

  • SSH3 shares homology with other Slingshot family members (SSH1, SSH2)

  • Validate antibody specificity against recombinant proteins of all family members

Methodological recommendation: When studying patient samples, use multiple antibodies targeting different SSH3 epitopes to ensure consistent findings and correlate with mRNA expression data where possible.

How should I select the appropriate SSH3 antibody for my experiment?

Selection of the optimal SSH3 antibody depends on several factors:

• Application requirements:

  • For Western blot: Both monoclonal and polyclonal antibodies work well

  • For IP/Co-IP: Validated antibodies with demonstrated ability to immunoprecipitate native protein are essential

  • For IHC/IF: Consider fixation compatibility and validated dilution ranges (1:50-1:500 for IHC, 1:200-1:800 for IF)

• Species compatibility:

  • Different antibodies have varying cross-reactivity profiles:

    • Human reactivity: All SSH3 antibodies in the search results

    • Mouse reactivity: Some rabbit polyclonal antibodies

    • Rat reactivity: Select antibodies

    • Predicted reactivity with other species: varies by antibody

• Epitope considerations:

  • N-terminal vs. C-terminal targeting may affect detection of specific isoforms

  • Phosphorylation-sensitive epitopes may affect detection under certain conditions

• Validation evidence:

  • Review published validation data (e.g., Western blot images, IHC results)

  • Consider antibodies with RRID identifiers and published citations

Decision matrix: For detecting total SSH3 across multiple applications, rabbit polyclonal antibodies offer versatility , while mouse monoclonal antibodies may provide higher specificity for particular applications or epitopes .

What controls should I include when validating an SSH3 antibody?

Proper antibody validation requires multiple controls:

• Positive controls:

  • Cell lines with confirmed SSH3 expression: HeLa, HT-29, A-431 cells

  • Tissues with known SSH3 expression: salivary gland, colon cancer, breast cancer

  • Recombinant SSH3 protein (useful for Western blot)

• Negative controls:

  • SSH3 knockdown/knockout cells (siRNA or CRISPR-modified)

  • Secondary antibody-only controls

  • Isotype controls (particularly for monoclonal antibodies)

• Specificity controls:

  • Pre-absorption with immunizing peptide to confirm specific binding

  • Comparison of staining patterns from two distinct SSH3 antibodies recognizing different epitopes

  • Cross-reactivity assessment with other Slingshot family proteins (SSH1, SSH2)

• Method-specific controls:

  • For Western blot: Molecular weight markers to confirm band size (90-95 kDa observed)

  • For IHC/IF: Subcellular localization consistent with known distribution (nuclear and cytoplasmic)

  • For IP: Non-specific IgG control to identify background binding

Methodological recommendation: Document and report all validation controls in publications to improve experimental reproducibility and antibody reliability assessment.

How should I interpret discrepancies in SSH3 detection between different antibodies?

When facing discrepancies between different SSH3 antibodies, consider these analysis strategies:

• Epitope mapping analysis:

  • Different antibodies target distinct epitopes that may be differentially accessible

  • Compare immunogen sequences between antibodies to identify potential differences

  • Epitopes near post-translational modification sites may show condition-dependent recognition

• Isoform-specific detection:

  • SSH3's five isoforms may be detected differently by antibodies targeting different regions

  • Compare results with mRNA expression analysis of specific isoforms

• Technical variables assessment:

  • Different antibodies have optimal protocols: adjust dilutions (1:1000-1:4000 for WB, 1:50-1:500 for IHC)

  • Compare results under multiple conditions (different fixatives, antigen retrieval methods)

• Cross-reactivity investigation:

  • Test antibodies on overexpression systems and knockdown/knockout samples

  • Consider possible cross-reactivity with SSH1 or SSH2 family members

When reporting discrepancies, document all experimental conditions and provide data from multiple antibodies to allow comprehensive interpretation.

What are the best practices for quantifying SSH3 expression in tissue samples?

For accurate quantification of SSH3 in tissues:

• Standardized IHC protocol optimization:

  • Follow validated protocols for antigen retrieval (TE buffer pH 9.0 or citrate buffer pH 6.0)

  • Use consistent antibody dilutions (typically 1:50-1:500)

  • Process all samples simultaneously when possible

• Scoring methodology:

  • Develop a consistent scoring system incorporating both staining intensity and percentage of positive cells

  • Use digital image analysis when possible for objective quantification

  • Account for both nuclear and cytoplasmic staining

• Multiple detection methods:

  • Correlate IHC findings with Western blot quantification from adjacent tissue

  • Consider complementary RNA-level analysis (qPCR, RNA-seq)

• Reference standards inclusion:

  • Include control tissues with known SSH3 expression levels

  • Use standardized positive controls across experiments

• Blinded assessment:

  • Have multiple observers score samples independently

  • Calculate inter-observer agreement statistics

Methodological recommendation: Report detailed quantification methodology, include representative images of scoring categories, and utilize statistical approaches appropriate for semi-quantitative data analysis.