ST5 Antibody

What is ST5 protein and why is it significant in cellular research?

ST5 (Suppression of Tumorigenicity 5) is a protein that plays a crucial role in cellular signaling pathways, particularly as a modulator of Ras signaling. The protein exists in multiple isoforms, with the long isoform functioning as a guanine nucleotide exchange factor (GEF) that can activate Ras/ERK signaling pathways in the presence of growth factors such as Epidermal Growth Factor (EGF) . Research has demonstrated that ST5 has a dual role in Ras signaling - it can function both as an inhibitor and activator depending on which isoform is expressed. In cellular localization studies, ST5 demonstrates both membrane and cytoplasmic signals, consistent with its role in Ras activation, as activated Ras is typically anchored to the cytoplasmic face of the plasma membrane .

What types of ST5 antibodies are available for research applications?

Multiple types of ST5 antibodies are available for research applications, varying in their epitope recognition, species reactivity, and conjugation status:

• Antibodies targeting different regions of ST5:

  • Internal region antibodies

  • N-terminal region antibodies (AA 3-280)

  • C-terminal region antibodies (AA 1106-1137)

  • Specific peptide region antibodies (e.g., AA 68-88, AA 387-436)

• Species reactivity profiles:

  • Human-specific antibodies

  • Human and mouse cross-reactive antibodies

  • Multi-species reactive antibodies (human, cow, guinea pig, horse, rabbit, dog, bat, monkey)

• Conjugation variants:

  • Unconjugated antibodies

  • FITC-conjugated antibodies for fluorescence applications

  • HRP-conjugated antibodies for enhanced detection

  • Biotin-conjugated antibodies for amplification strategies

Most ST5 antibodies currently available are polyclonal, typically developed in rabbit hosts, and have been validated for applications including Western Blotting, ELISA, immunohistochemistry, and immunofluorescence .

How can researchers validate the specificity of ST5 antibodies?

Validating antibody specificity is crucial for ensuring reliable experimental results. For ST5 antibodies, the following validation approaches are recommended:

• Genetic approaches:

  • siRNA or shRNA knockdown: Demonstrate reduced signal intensity corresponding to ST5 knockdown efficiency

  • CRISPR/Cas9 knockout: Show complete absence of signal in knockout cells/tissues

  • Overexpression: Demonstrate increased signal intensity proportional to overexpression levels

  • Use of the doxycycline-inducible system: Show signal intensity correlating with induction levels

• Biochemical approaches:

  • Peptide competition assay: Pre-incubation of antibody with immunizing peptide should abolish specific signal

  • Immunoprecipitation followed by mass spectrometry: Confirm identity of pulled-down protein

  • Use of multiple antibodies targeting different epitopes: Consistent results across antibodies increase confidence

• Application-specific validations:

  • For Western blotting: Confirm band at expected molecular weight for the specific isoform

  • For IHC/ICC: Compare with RNA expression data or known expression patterns

  • For ELISA: Establish standard curves with recombinant protein and determine detection limits

ST5 polyclonal antibodies are typically affinity-purified from rabbit antiserum by affinity-chromatography using epitope-specific immunogen, which enhances their specificity . Researchers should remember that validation should be performed for each specific application, as an antibody validated for Western blotting may not necessarily work in immunofluorescence or other applications.

How can ST5 antibodies be utilized in studying Ras/ERK signaling pathways?

ST5 antibodies serve as valuable tools for investigating Ras/ERK signaling pathways through multiple experimental approaches:

• Protein expression analysis: Western blotting with ST5 antibodies enables quantification of ST5 protein levels in different cellular contexts, allowing researchers to correlate ST5 expression with Ras/ERK pathway activation states .

• Co-immunoprecipitation studies: ST5 antibodies can be used to pull down ST5 protein complexes, helping identify binding partners and regulatory molecules in the Ras/ERK signaling cascade.

• Subcellular localization studies: Immunofluorescence techniques utilizing ST5 antibodies can reveal the dynamic localization of ST5 in response to growth factor stimulation, providing insights into its mechanism of action in Ras activation .

• Functional validation: ST5 antibodies can verify the efficacy of genetic manipulation approaches (overexpression, knockdown) by confirming altered protein expression, validating observed phenotypic effects .

When designing experiments to study ST5's role in Ras/ERK signaling, researchers should consider using positive controls such as EGF stimulation, which has been shown to enhance ST5-mediated Ras activation. Additionally, examining downstream phosphorylation of ERK (pERK) serves as a functional readout of pathway activation .

What role does ST5 play in β-cell proliferation and diabetes research?

The role of ST5 in β-cell proliferation and diabetes research has been investigated using genetic models. According to research findings, investigators hypothesized that ST5 overexpression might drive adult β-cell proliferation due to its role in activating MAPK/ERK signaling pathways, which are required for augmented β-cell proliferation during pregnancy .

To test this hypothesis, researchers developed a doxycycline-inducible bitransgenic mouse model that allowed β-cell-specific expression of human ST5. The system utilized a Tetracycline Response Element (TRE) controlling ST5 expression and Rat Insulin Promoter (RIP) driving expression of reverse tetracycline-controlled transactivator (rtTA) .

Key findings from this research include:

• Successful induction of ST5 expression in β-cells, confirmed by strong membrane and cytoplasmic signals consistent with ST5's role in Ras activation

• ST5 overexpression did not adversely affect β-cell identity, as evidenced by maintained insulin and Pdx1 protein expression

• Contrary to expectations, ST5 overexpression alone did not increase β-cell proliferation under basal conditions

• ST5 overexpression did not enhance β-cell proliferation during metabolically challenged states (pregnancy or streptozotocin-induced β-cell loss)

• Glucose tolerance remained comparable between ST5-overexpressing and control mice

These findings suggest that while ST5 can activate Ras/ERK signaling, additional factors may be required for promoting β-cell proliferation, highlighting the complexity of signaling networks governing β-cell mass expansion .

How can ST5 antibodies be used in the development of monoclonal antibody production techniques?

ST5 has been utilized in innovative approaches for immortalizing B cells to produce monoclonal antibodies. Research has demonstrated that expressing an inducible active mutant of the transcription factor Signal Transducer and Activator of Transcription 5 (STAT5) in human memory B cells can inhibit their differentiation while increasing their replicative lifespan .

This methodology involved:

• Isolating memory B cells from healthy donors

• Introducing an inducible active STAT5 mutant

• Establishing cloned B cell lines with the capacity to undergo plasma cell differentiation

• Turning off STAT5 expression to induce immunoglobulin secretion

• Screening the resulting clones for antigen-specific antibody production

The researchers successfully applied this method to obtain monoclonal antibodies against tetanus toxoid. The approach offers several advantages over traditional hybridoma techniques:

• It utilizes human B cells rather than mouse cells, avoiding species incompatibility issues

• The method can potentially capture the full diversity of the human antibody repertoire

• The conditional expression system allows for controlled antibody production

• Selected antibodies have demonstrated functionality in both binding assays and in vivo challenge models

This innovative use of STAT5 in monoclonal antibody development represents a valuable advancement in antibody engineering and production methodology, with potential applications for generating therapeutic antibodies against various targets .

What are the optimal protocols for using ST5 antibodies in Western Blotting?

Optimal protocols for using ST5 antibodies in Western Blotting involve several critical steps that must be carefully optimized:

• Sample preparation:

  • Lyse cells in a buffer containing protease inhibitors to prevent degradation

  • For membrane-associated proteins like ST5, include appropriate detergents

  • Normalize protein concentration (typically 20-50 μg total protein per lane)

  • Denature samples in Laemmli buffer with reducing agent

• Gel electrophoresis and transfer:

  • Use appropriate percentage polyacrylamide gels for optimal resolution of ST5 isoforms

  • Transfer to PVDF or nitrocellulose membrane using standard protocols

  • Verify transfer efficiency with reversible protein stains

• Blocking and antibody incubation:

  • Block membrane in 5% non-fat dry milk or 5% BSA in TBST

  • Dilute primary ST5 antibody according to manufacturer's recommendation (typically 1:500 to 1:2000)

  • Incubate with primary antibody overnight at 4°C with gentle rocking

  • Wash thoroughly with TBST

  • Incubate with appropriate HRP-conjugated secondary antibody

  • Wash thoroughly with TBST

• Detection and analysis:

  • Develop using enhanced chemiluminescence (ECL) substrate

  • Use appropriate loading controls for normalization

  • Quantify band intensity using image analysis software

Available ST5 antibodies have been validated for Western blotting applications and can detect endogenous levels of ST5 protein . These antibodies have demonstrated the ability to recognize ST5 in human and mouse samples, making them versatile tools for comparative studies across species .

What factors should be considered when using ST5 antibodies for immunofluorescence?

When using ST5 antibodies for immunofluorescence applications, researchers should consider:

• Fixation and permeabilization:

  • Optimize fixation method (paraformaldehyde, methanol, or acetone) based on epitope sensitivity

  • Select appropriate permeabilization agents (Triton X-100, saponin, or digitonin) depending on the subcellular localization of ST5

  • Consider antigen retrieval methods if signal is weak

• Antibody selection and optimization:

  • Choose ST5 antibodies specifically validated for immunofluorescence

  • Test a range of antibody dilutions to determine optimal signal-to-noise ratio

  • Consider using antibodies that target the internal region of ST5 for better accessibility

• Controls and validation:

  • Include positive controls (cells/tissues known to express ST5)

  • Use negative controls (secondary antibody only, isotype controls)

  • Validate specificity through knockdown/overexpression approaches

  • Consider counterstaining with markers of subcellular compartments to confirm localization

• Signal detection and analysis:

  • Select appropriate fluorophore-conjugated secondary antibodies or directly conjugated primaries (FITC-conjugated ST5 antibodies are available)

  • Optimize imaging parameters (exposure time, gain settings)

  • Use appropriate filter sets to minimize bleed-through

  • Consider confocal microscopy for detailed subcellular localization studies

ST5 typically shows both membrane and cytoplasmic localization patterns, consistent with its role in Ras activation . This localization pattern can serve as an internal validation when assessing antibody performance in immunofluorescence applications.

How should researchers approach quantitative analysis of ST5 expression?

Quantitative analysis of ST5 expression requires careful experimental design and appropriate analytical approaches:

• Western blotting quantification:

  • Use appropriate loading controls (β-actin, GAPDH) for normalization

  • Ensure signal is within linear range of detection

  • Analyze multiple biological replicates

  • Use image analysis software with background subtraction

  • Present data as fold-change relative to control conditions

• Immunofluorescence quantification:

  • Standardize image acquisition parameters across all samples

  • Use automated analysis algorithms for unbiased quantification

  • Measure integrated density or mean fluorescence intensity

  • Analyze multiple fields and cells for statistical robustness

  • Consider subcellular distribution patterns

• ELISA-based quantification:

  • Several ST5 ELISA kits are available for quantitative analysis

  • Generate standard curves using recombinant ST5 protein

  • Ensure samples fall within the linear range of the assay

  • Test dilution linearity to confirm specificity

  • Include technical and biological replicates

• Flow cytometry quantification:

  • Use appropriate ST5 antibodies validated for flow cytometry applications

  • Include fluorescence-minus-one (FMO) controls

  • Calculate median fluorescence intensity rather than mean for non-normal distributions

  • Consider multiparameter analysis with other markers

• Data analysis considerations:

  • Apply appropriate statistical tests based on data distribution

  • Account for multiple comparisons when analyzing different conditions

  • Consider normalized rather than absolute expression values for comparisons across experiments

  • Present both representative images/blots and quantitative data

By following these methodological guidelines, researchers can generate reliable quantitative data on ST5 expression across different experimental conditions and cell types.

What are common challenges when working with ST5 antibodies?

Researchers working with ST5 antibodies may encounter several common challenges:

• Isoform specificity issues:

  • ST5 exists in multiple isoforms (including a long form with activating properties and shorter forms)

  • Some antibodies may recognize specific isoforms while others detect all variants

  • This can lead to unexpected banding patterns in Western blots

  • Solution: Carefully review antibody epitope information and design experiments accordingly

• Low endogenous expression:

  • Some cell types may express low levels of ST5, making detection challenging

  • Solution: Optimize protein loading amounts, use more sensitive detection methods, or consider signal amplification techniques

• Cross-reactivity with related proteins:

  • ST5 shares sequence homology with other proteins

  • Solution: Validate specificity through knockout/knockdown experiments and multiple detection methods

• Lot-to-lot variability:

  • Especially common with polyclonal antibodies

  • Solution: Purchase larger lots for long-term studies or validate new lots against previous ones

• Application-specific optimization requirements:

  • An antibody that works well in Western blotting may fail in IHC or IF

  • Solution: Select antibodies specifically validated for your application of interest

• Species-specific considerations:

  • Many ST5 antibodies show limited cross-reactivity across species

  • Solution: Select antibodies validated for your species of interest (human, mouse, or multi-species reactive options are available)

How can researchers minimize non-specific binding when using ST5 antibodies?

Non-specific binding can significantly impact the quality and reliability of results when working with ST5 antibodies. Here are strategies to minimize non-specific binding:

• Blocking optimization:

  • Test different blocking agents (BSA, non-fat dry milk, normal serum, commercial blockers)

  • Adjust blocking time and concentration

  • For Western blots, match the blocking agent with the diluent used for antibodies

• Antibody dilution optimization:

  • Titrate antibody concentrations to determine optimal signal-to-noise ratio

  • Generally, using the highest dilution that provides specific signal helps reduce background

  • Follow manufacturer's recommended dilutions as starting points

• Buffer modifications:

  • Add appropriate detergents to reduce hydrophobic interactions

  • Include sufficient salt to reduce ionic interactions

  • Consider adding carrier proteins to reduce non-specific binding

• Sample preparation considerations:

  • Ensure complete lysis and denaturation for Western blotting

  • Optimize fixation and permeabilization for immunofluorescence

  • Remove lipids and other interfering substances that may cause non-specific binding

• Antibody quality:

  • Use affinity-purified antibodies when available (such as the ST5 polyclonal antibodies purified by affinity-chromatography using epitope-specific immunogen)

  • Consider antibodies validated for your specific application

• Wash protocol optimization:

  • Increase number and duration of washes

  • Use appropriate detergent concentration in wash buffers

  • Ensure complete buffer removal between wash steps

What strategies can be employed when ST5 antibodies yield inconsistent results?

When ST5 antibodies yield inconsistent results, researchers can employ several strategies:

• Antibody validation and selection:

  • Test multiple antibodies targeting different epitopes of ST5

  • Verify antibody performance using positive controls (cells with known ST5 expression)

  • Consider switching from polyclonal to monoclonal antibodies for greater consistency

• Protocol standardization:

  • Implement highly detailed, step-by-step protocols

  • Control critical variables (timing, temperature, reagent concentrations)

  • Use automated systems where possible to reduce operator variability

• Sample handling optimization:

  • Standardize sample collection and processing

  • Minimize freeze-thaw cycles of both samples and antibodies

  • Prepare fresh working solutions of antibodies for each experiment

• Alternative detection approaches:

  • If one application (e.g., Western blotting) yields inconsistent results, validate findings using complementary methods (e.g., ELISA, immunofluorescence)

  • Consider using recombinant tagged ST5 as a positive control

• Biological validation:

  • Correlate antibody results with mRNA expression data

  • Use genetic manipulation (overexpression, knockdown) to confirm specificity

  • Consider the doxycycline-inducible system described for ST5 expression as a validation tool

• Technical replication:

  • Increase number of technical replicates

  • Perform experiments on different days

  • Have multiple researchers follow the same protocol independently

By systematically addressing these aspects, researchers can improve the consistency and reliability of results obtained with ST5 antibodies.

How should researchers interpret ST5 expression patterns across different cell types?

Interpreting ST5 expression patterns across different cell types requires consideration of multiple biological and technical factors:

• Biological explanations for expression variation:

  • Cell type-specific expression levels of ST5 protein

  • Differential expression of ST5 isoforms in different tissues

  • Post-translational modifications affecting epitope accessibility

  • Protein-protein interactions modulating ST5 detection

  • Subcellular localization differences (membrane vs. cytoplasmic distribution)

• Technical considerations for interpretation:

  • Normalize to appropriate housekeeping proteins

  • Consider relative rather than absolute expression comparisons

  • Validate with orthogonal methods (qPCR, mass spectrometry)

  • Use multiple antibodies targeting different epitopes of ST5

  • Include positive control cell lines with known ST5 expression

• Functional context analysis:

  • Correlate ST5 expression with Ras/ERK pathway activation markers

  • Consider the relationship between ST5 expression and cellular phenotypes

  • Integrate with knowledge about cell type-specific signaling networks

  • Examine relationship to known ST5 regulators in different cell types

Some ST5 antibodies demonstrate reactivity across multiple species (human, mouse) and some are reactive across an even wider range (human, cow, guinea pig, horse, rabbit, dog, bat, monkey) , enabling comparative studies across species. This cross-reactivity should be considered when interpreting expression patterns in different model systems.

What can be learned from ST5 localization patterns in cells?

ST5 localization patterns can provide valuable insights into its function and regulation:

• Membrane localization implications:

  • ST5 demonstrates membrane localization consistent with its role in Ras activation

  • Membrane-associated ST5 likely interacts with membrane-bound Ras proteins

  • Changes in membrane association may reflect activation states or post-translational modifications

  • Co-localization with specific membrane domains may indicate functional interactions

• Cytoplasmic localization considerations:

  • Cytoplasmic ST5 signals are also observed in cells

  • May represent inactive pools or different functional states

  • Could indicate interactions with cytoplasmic signaling components

  • Changes in cytoplasmic distribution might reflect responses to stimuli

• Dynamic regulation assessment:

  • Changes in ST5 localization following growth factor stimulation may indicate activation

  • Redistribution between cellular compartments might reflect regulatory mechanisms

  • Temporal changes in localization patterns may correlate with signaling dynamics

• Co-localization analysis:

  • Co-localization with Ras family proteins would support functional interaction

  • Association with endosomal markers might indicate trafficking or regulation

  • Co-localization with other signaling components could reveal novel interactions

• Methodological approaches:

  • Use confocal microscopy for precise subcellular localization

  • Consider super-resolution techniques for detailed analysis

  • Employ fluorescently tagged ST5 for live-cell imaging of dynamics

  • Use appropriate compartment markers for co-localization studies

Understanding ST5 localization patterns can provide mechanistic insights into how it functions in different cellular contexts and how its activity might be regulated through subcellular targeting.

How can ST5 antibody data be integrated with other molecular markers in multi-parameter analyses?

Integrating ST5 antibody data with other molecular markers enhances the depth and reliability of research findings:

• Multi-parameter analysis approaches:

  • Multiplex immunofluorescence/immunohistochemistry:

    • Co-staining ST5 with Ras pathway components (Ras, pERK)

    • Cell type-specific markers to assess expression patterns

    • Subcellular compartment markers to evaluate localization

  • Flow cytometry with multiple markers

  • Sequential immunoblotting on the same membrane

• Correlation analyses:

  • Quantitative comparison of ST5 levels with:

    • Ras/ERK pathway activation markers

    • Cell proliferation markers (Ki67, BrdU incorporation)

    • Cell differentiation markers

  • Statistical correlation methods for identifying relationships

  • Multivariate analysis techniques for complex datasets

• Functional integration strategies:

  • Phospho-protein analysis in parallel with ST5 detection

  • Activity assays for related signaling molecules

  • Genetic manipulation of ST5 followed by molecular profiling

  • Pharmacological intervention in ST5-related pathways

• Multi-omics integration:

  • Correlating protein expression (ST5) with transcriptomic data

  • Integrating with proteomic analyses for network identification

  • Pathway enrichment analysis incorporating ST5 data

By integrating ST5 antibody data with complementary molecular markers, researchers can develop a more comprehensive understanding of ST5's role in biological processes and its relationships with other signaling components, particularly in contexts such as β-cell biology and B-cell function .