STK4 Antibody, Biotin conjugated

What is the functional significance of STK4 in cellular signaling pathways?

STK4 is a stress-activated, pro-apoptotic kinase that plays a pivotal role in the Hippo signaling pathway. Following caspase-cleavage, STK4 enters the nucleus and induces chromatin condensation followed by internucleosomal DNA fragmentation . As a key component of the Hippo pathway, STK4 restricts proliferation and promotes apoptosis, thereby contributing to organ size control and tumor suppression .

The core Hippo pathway consists of a kinase cascade where STK4/MST1 and STK3/MST2, in complex with the regulatory protein SAV1, phosphorylate and activate LATS1/2 in complex with its regulatory protein MOB1. This activation leads to phosphorylation and inactivation of the YAP1 oncoprotein and WWTR1/TAZ, preventing their translocation to the nucleus and subsequent regulation of genes involved in cell proliferation, death, and migration .

What are the recommended applications for biotin-conjugated STK4 antibodies?

Biotin-conjugated STK4 antibodies are versatile research tools applicable to multiple experimental techniques:

When using these antibodies, it's crucial to optimize dilutions for specific experimental conditions. The biotin conjugation provides enhanced sensitivity for detection systems utilizing streptavidin-based reagents .

What reactive species can STK4 antibodies detect, and how does this impact experimental design?

When selecting an STK4 antibody for your research, consider the species reactivity carefully:

Species cross-reactivity is critical when designing experiments using animal models. For example, if you're conducting comparative studies between human and mouse samples, ensure your antibody has confirmed reactivity with both species. For evolutionary studies or when working with less common model organisms, select antibodies with broader predicted reactivity ranges .

How should researchers evaluate STK4 antibody specificity and validate experimental results?

Antibody validation is crucial for ensuring reliable results in STK4 research:

• Western blot validation: Verify a single band at the expected molecular weight (~55.63 kDa for full-length STK4) . Note that multiple bands may appear if detecting both full-length and cleaved forms (37 kDa N-terminal and 18 kDa C-terminal fragments) .

• Positive and negative controls: Include cell lines known to express STK4 (like HeLa cells) as positive controls . For negative controls, use cells with siRNA-mediated knockdown of STK4, which should show significantly reduced signal .

• Cross-validation: Employ multiple detection methods (e.g., WB, IHC, and IF) to confirm consistent STK4 expression patterns .

• Subcellular localization: Confirm proper localization pattern - STK4 should be primarily cytoplasmic in resting cells but can translocate to the nucleus during apoptosis .

• Functional validation: In studies examining STK4's role in the Hippo pathway, validate by demonstrating expected downstream effects, such as changes in LATS1/2 phosphorylation or YAP1 localization .

What are the critical methodological considerations when working with biotin-conjugated STK4 antibodies?

Working with biotin-conjugated antibodies requires specific methodological attention:

• Endogenous biotin interference: Tissues with high endogenous biotin levels (kidney, liver, brain) may produce background signal. Block endogenous biotin using avidin/biotin blocking kits before antibody application .

• Storage and stability: Store biotin-conjugated STK4 antibodies at -20°C for optimal stability (up to 12 months). Avoid repeated freeze-thaw cycles, which can degrade both the antibody and the biotin conjugate .

• Detection systems: Use streptavidin-conjugated enzymes or fluorophores with high affinity for biotin. The avidin-biotin complex (ABC) method offers signal amplification advantages for low abundance proteins .

• Buffer considerations: Use antibody stabilization buffers containing BSA (typically 1%) and glycerol (typically 50%) to maintain antibody activity. Avoid buffers that might interfere with the biotin-streptavidin interaction .

• Biotin blocking in multiplex assays: When performing multiplex staining with multiple biotin-conjugated antibodies, complete each detection step sequentially with blocking between steps to prevent cross-reactivity .

How can researchers effectively analyze STK4 expression patterns in different disease contexts?

STK4 expression analysis requires careful consideration of tissue and disease-specific contexts:

• Cancer tissue analysis: STK4 expression patterns vary across cancer types. In endometrial cancer, STK4 expression is generally low at both mRNA and protein levels, particularly in serous tumors. Interestingly, higher STK4 expression correlates with worse prognosis in serous endometrial cancer but shows no such relationship in endometrioid endometrial cancer .

• Quantification methods: For IHC studies, use digital image analysis to quantify staining intensity and percentage of positive cells. Semi-quantitative scoring systems (0-3+) can be employed, but automated analysis provides more objective results .

• Correlation with clinical data: Always correlate STK4 expression with clinicopathological data including tumor grade, stage, and patient survival to identify prognostic value .

• Subcellular localization: Distinguish between cytoplasmic and nuclear STK4 staining, as translocation patterns can provide insights into activation status and functional implications .

• Multi-omics approach: Combine protein-level data (from antibody-based methods) with mRNA expression analysis to identify potential post-transcriptional regulatory mechanisms affecting STK4 expression .

How can STK4 antibodies be used to investigate STK4 deficiency-related immunological disorders?

STK4 deficiency is associated with a rare, autosomal recessive primary immunodeficiency syndrome. Research approaches include:

• Protein expression analysis: Use STK4 antibodies to confirm protein deficiency in patient samples. Western blot analysis can detect complete absence or reduced levels of STK4 protein in peripheral blood mononuclear cells (PBMCs) .

• Functional assays: Investigate downstream effects of STK4 deficiency by examining:

  • T cell subset abnormalities

  • Increased T cell apoptosis

  • Impaired interferon signaling

  • Altered cytokine-induced adhesion genes

• Genotype-phenotype correlations: Correlate specific STK4 mutations with protein expression levels and clinical presentations. Novel mutations can be verified by showing their impact on protein expression using antibody-based techniques .

• Treatment monitoring: STK4 antibodies can help monitor protein expression restoration following interventions or gene therapy approaches .

• Co-immunoprecipitation studies: Use STK4 antibodies for co-IP to investigate altered protein-protein interactions in STK4-deficient cells, particularly focusing on Hippo pathway components and FoxO proteins .

What role does STK4 play in cancer development and how can STK4 antibodies contribute to cancer research?

STK4 functions as a tumor suppressor through the Hippo pathway, making it relevant to cancer research:

• Expression analysis in tumor samples: STK4 antibodies can be used to assess expression levels across cancer types and stages. For instance, in endometrial cancer, STK4 expression patterns differ between histological subtypes and correlate with prognosis .

• Functional studies: Investigate STK4's role in:

  • Restricting cell proliferation

  • Promoting apoptosis

  • Regulating tumor suppressor genes

  • Controlling organ size

• Pathway interactions: STK4 antibodies can help elucidate interactions between STK4 and other cancer-related pathways:

  • p53-dependent transcription and apoptosis (STK4 phosphorylates SIRT1 and inhibits SIRT1-mediated p53 deacetylation)

  • PKB/AKT1 signaling (STK4 acts as an inhibitor)

  • Androgen receptor (AR) signaling (STK4 phosphorylates AR on Ser-650 and suppresses its activity)

• Prognostic markers: Evaluate STK4 as a prognostic marker in specific cancer types. For example, in serous endometrial cancer, higher STK4 expression correlates with worse prognosis .

How can researchers optimize immunohistochemistry protocols for biotin-conjugated STK4 antibodies?

Effective IHC with biotin-conjugated STK4 antibodies requires attention to specific protocol details:

• Antigen retrieval optimization: Test both heat-induced epitope retrieval (HIER) methods using citrate buffer (pH 6.0) and EDTA buffer (pH 9.0) to determine optimal conditions for STK4 epitope exposure .

• Blocking endogenous biotin: Tissue samples, especially liver, kidney, and brain, contain high levels of endogenous biotin that can cause background staining. Implement an avidin-biotin blocking step before antibody application .

• Antibody titration: Perform a dilution series (typically 1:50 to 1:400) to determine optimal antibody concentration that maximizes specific signal while minimizing background .

• Detection system selection: While ABC (avidin-biotin complex) methods provide signal amplification, they may increase background. For tissues with high STK4 expression, consider using streptavidin-HRP directly for cleaner results .

• Counterstaining optimization: Since STK4 can be both cytoplasmic and nuclear, optimize counterstaining intensity to ensure accurate visualization of subcellular localization .

• Positive and negative controls: Include known STK4-positive tissues and matched tissues treated with isotype control antibodies. For definitive negative controls, consider using STK4-deficient patient samples if available .

What considerations should be made when designing flow cytometry experiments with biotin-conjugated STK4 antibodies?

Flow cytometry with biotin-conjugated STK4 antibodies requires specific optimization:

• Cell fixation and permeabilization: STK4 is primarily intracellular, requiring effective permeabilization. Test commercial permeabilization kits specifically designed for intracellular kinases .

• Titration for optimal signal-to-noise ratio: Conduct antibody titration experiments (typically starting at 1:50-1:200) to determine the concentration providing maximum separation between positive and negative populations .

• Multi-color panel design: When incorporating biotin-conjugated STK4 antibodies into multi-color panels, use streptavidin conjugated to fluorophores that minimize spectral overlap with other markers in your panel.

• Controls for biotin-based detection: Include FMO (fluorescence minus one) controls with streptavidin-fluorophore alone to account for any non-specific binding of the detection reagent .

• Gating strategy: When analyzing STK4 expression in immune cell subsets, establish a clear gating strategy that accounts for potential differences in autofluorescence between cell populations.

• Quantification approaches: Consider using molecules of equivalent soluble fluorochrome (MESF) beads for standardization when comparing STK4 expression levels between experiments or study subjects.

How can researchers incorporate biotin-conjugated STK4 antibodies in studying the Hippo signaling pathway dynamics?

The Hippo pathway is a complex signaling cascade with STK4 as a key component. Research approaches include:

• Co-immunoprecipitation studies: Use biotin-conjugated STK4 antibodies to pull down STK4 and its interaction partners (SAV1, LATS1/2, MOB1) followed by streptavidin-based purification for higher specificity than traditional IP approaches .

• Proximity ligation assays (PLA): Combine biotin-conjugated STK4 antibodies with antibodies against other Hippo pathway components to visualize and quantify protein-protein interactions in situ with high sensitivity.

• Tissue microarray analysis: Apply biotin-conjugated STK4 antibodies to tissue microarrays to efficiently evaluate STK4 expression across multiple tissue samples and correlate with other Hippo pathway components .

• Activation state-specific detection: Develop assays that distinguish between full-length (inactive) and cleaved (active) forms of STK4 using antibodies that recognize different epitopes, allowing for assessment of pathway activation status .

• Multiplexed IF imaging: Combine biotin-conjugated STK4 antibodies with antibodies against downstream targets like phosphorylated LATS1/2 and YAP/TAZ localization to visualize pathway activation in single cells.

• Time-course experiments: Utilize biotin-conjugated STK4 antibodies in time-course experiments following pathway stimulation to track dynamic changes in STK4 localization and activation state.

What are common issues encountered when using biotin-conjugated STK4 antibodies and how can they be resolved?

Researchers may encounter several challenges when working with biotin-conjugated STK4 antibodies:

• High background signal:

  • Cause: Endogenous biotin in tissues or insufficient blocking

  • Solution: Implement avidin-biotin blocking steps before antibody application; increase blocking time/concentration; reduce antibody concentration

• Weak or absent signal:

  • Cause: Insufficient antigen retrieval, antibody degradation, or low STK4 expression

  • Solution: Optimize antigen retrieval conditions; verify antibody integrity with a positive control; consider signal amplification systems

• Non-specific binding:

  • Cause: Excessive antibody concentration or cross-reactivity

  • Solution: Titrate antibody; include additional washing steps; validate with knockout/knockdown controls

• Inconsistent results between experiments:

  • Cause: Variations in blocking, incubation times, or detection systems

  • Solution: Standardize protocols; use automated systems when possible; implement positive controls in each experiment

• Discrepancies between protein and mRNA levels:

  • Cause: Post-transcriptional regulation of STK4

  • Solution: Combine antibody-based protein detection with mRNA analysis to gain comprehensive understanding

How should researchers interpret conflicting results regarding STK4 expression and function?

STK4 research sometimes yields apparently contradictory results due to context-dependent functions:

• Dual role in apoptosis: STK4 has been described as both pro-apoptotic and anti-apoptotic. This apparent contradiction stems from context-dependent functions:

  • Pro-apoptotic: STK4 is cleaved by caspases, and the N-terminal fragment translocates to the nucleus to promote apoptosis

  • Anti-apoptotic: STK4 phosphorylates FoxO proteins, which can protect against oxidative stress-induced cell death

• Tissue-specific expression patterns: STK4 expression and function vary across tissues:

  • In immune cells: STK4 deficiency leads to lymphopenia and increased susceptibility to apoptosis

  • In cancer: STK4's role may be context-dependent, with expression patterns differing between cancer types and stages

• Methodological variations: Different antibody clones and detection methods may yield varying results:

  • Antibody epitope location matters: Some antibodies detect only full-length STK4, while others detect both full-length and cleaved forms

  • Detection method sensitivity: Techniques like Western blot and IHC may yield different results due to differences in sensitivity and ability to detect spatial distribution

• Integration approach: When facing conflicting data:

  • Employ multiple antibodies targeting different epitopes

  • Use multiple detection techniques (WB, IHC, IF, flow cytometry)

  • Correlate with functional assays measuring downstream pathway activation

  • Consider genetic approaches (knockdown/knockout) to validate antibody specificity

How might biotin-conjugated STK4 antibodies be utilized in single-cell analysis techniques?

Single-cell technologies offer new opportunities for studying STK4 biology:

• Single-cell Western blotting: Biotin-conjugated STK4 antibodies can be applied to microfluidic single-cell Western blotting platforms to analyze STK4 expression heterogeneity within seemingly homogeneous cell populations.

• Mass cytometry (CyTOF): Although not directly using biotin conjugation, antibodies validated with biotin-conjugated versions can be metal-tagged for high-parameter analysis of STK4 expression alongside dozens of other proteins at single-cell resolution.

• Spatial transcriptomics correlation: Combine IHC using biotin-conjugated STK4 antibodies with spatial transcriptomics to correlate protein expression with transcriptional profiles in tissue microenvironments.

• In situ proximity ligation assay (PLA): Use biotin-conjugated STK4 antibodies in combination with antibodies against interaction partners to visualize protein complexes in individual cells.

• Imaging mass cytometry: Apply metal-tagged versions of validated STK4 antibodies for highly multiplexed imaging of tissues with subcellular resolution.

What are the implications of recent STK4 research for therapeutic development, and how can antibodies contribute?

STK4's roles in immune function and cancer make it a potential therapeutic target:

• Target validation: Biotin-conjugated STK4 antibodies are essential for validating STK4 as a therapeutic target by:

  • Confirming expression in disease tissues

  • Elucidating mechanism of action

  • Identifying patient populations likely to benefit from STK4-targeted therapies

• Biomarker development: STK4 expression patterns may serve as biomarkers for:

  • Cancer prognosis (e.g., in serous endometrial cancer)

  • Immunodeficiency diagnosis and classification

  • Treatment response prediction

• Therapeutic strategies:

  • For STK4 deficiency: Gene therapy approaches could restore STK4 function in immunodeficient patients

  • For cancer: Modulating STK4 activity could potentially enhance tumor suppression through the Hippo pathway

• Companion diagnostics: If STK4-targeted therapies are developed, biotin-conjugated STK4 antibodies could form the basis of companion diagnostic assays to identify suitable patients.

• Monitoring therapy effects: Antibody-based assays can track changes in STK4 expression or activation state during treatment, providing valuable pharmacodynamic information.

What are the detailed specifications of commercially available biotin-conjugated STK4 antibodies?

How do experimental conditions affect the performance of biotin-conjugated STK4 antibodies?

Various experimental factors can influence antibody performance:

• Fixation methods:

  • Formalin fixation: May mask epitopes, requiring optimized antigen retrieval

  • Methanol fixation: Often preserves protein antigens but can affect tissue morphology

  • Paraformaldehyde: Commonly used for immunofluorescence with good epitope preservation

• Buffer and pH conditions:

  • For Western blotting: TBST (pH 7.4-7.6) with 5% non-fat milk or BSA typically works well

  • For IHC: Citrate buffer (pH 6.0) often provides good antigen retrieval for STK4

  • For immunofluorescence: PBS (pH 7.4) with 1% BSA is commonly effective

• Incubation parameters:

  • Temperature: 4°C overnight incubation may provide better signal-to-noise ratio than shorter room temperature incubations

  • Antibody concentration: Lower concentrations (higher dilutions) generally provide better specificity but may require signal amplification

• Detection systems:

  • ABC method: Provides signal amplification but may increase background

  • Streptavidin-enzyme direct conjugates: May offer cleaner results with less background

  • Fluorescent streptavidin conjugates: Allow for multiplexed detection and subcellular localization studies

• Sample preparation:

  • Fresh vs. frozen vs. FFPE tissue: Each requires different optimization strategies

  • Cell lines vs. primary cells: Expression levels and localization patterns may differ

  • Protein extraction methods: Affect protein yield and preservation of native conformation