STK25 Antibody

What is STK25 and why is it a target of research interest?

STK25 (serine/threonine kinase 25), also known as SOK1, YSK1, or Ste20 yeast homolog, is a protein kinase with a molecular mass of approximately 48.1 kilodaltons in humans . This kinase has gained significant research attention due to its involvement in multiple cellular pathways including metabolism, cell polarization, and stress responses. STK25 has been implicated in regulating lipid accumulation and metabolism in liver and adipose tissue, making it a potential therapeutic target for metabolic disorders . Additionally, recent research has demonstrated STK25's role in the Hippo signaling pathway through its interactions with SAV1-STRIPAK complexes, highlighting its importance in growth control and organ size regulation . The diverse functions of STK25 across different tissues and cellular processes have sparked interest in developing specific antibodies to study its expression, localization, and activity in various experimental settings.

What applications are STK25 antibodies commonly used for in research?

STK25 antibodies are employed in a wide range of experimental applications to investigate the protein's expression, localization, and function. Western blotting (WB) is the most frequently utilized technique for detecting STK25 protein levels in cell or tissue lysates, with validated antibodies being crucial for obtaining specific signals . Immunohistochemistry on paraffin-embedded sections (IHC-p) allows researchers to examine STK25 localization within tissue architecture, while immunocytochemistry (ICC) and immunofluorescence (IF) techniques provide cellular and subcellular localization information in cultured cells . These applications have been instrumental in identifying STK25's association with lipid droplets in hepatocytes and its presence in various subcellular compartments . When selecting an STK25 antibody, researchers should consider the specific application requirements, including the species reactivity needed (human, mouse, rat, or other orthologs), antibody type (monoclonal vs. polyclonal), and the epitope region targeted by the antibody, as these factors significantly impact experimental outcomes and data interpretation.

How should STK25 antibodies be validated before experimental use?

Proper validation of STK25 antibodies is essential to ensure experimental reliability and reproducibility. The gold standard for antibody validation involves testing the antibody in samples from knockout models, as exemplified by studies using Stk25−/− mice to validate anti-STK25 antibody specificity . This approach definitively confirms antibody specificity by demonstrating absence of signal in knockout tissues. For laboratories without access to knockout models, alternative validation strategies include siRNA-mediated knockdown of STK25 in cell culture, where researchers should observe a corresponding reduction in signal intensity proportional to the knockdown efficiency . Western blotting should yield a single band at the expected molecular weight (approximately 48.1 kDa for human STK25), while immunostaining techniques should show patterns consistent with known subcellular localizations of STK25. Cross-reactivity testing against related GCKIII family kinases (MST3 and MST4) is particularly important when studying STK25-specific functions, as these proteins share significant sequence homology and potentially overlapping functions . Validation data should be thoroughly documented, including positive and negative controls, to support the reliability of subsequent experimental findings.

What are the key considerations for Western blot protocols using STK25 antibodies?

Optimizing Western blot protocols for STK25 detection requires attention to several critical factors. Sample preparation is a crucial first step, with researchers typically using lysis buffers containing appropriate protease and phosphatase inhibitors to preserve both total STK25 and its phosphorylated forms . The selection of protein separation conditions significantly impacts detection quality, with 10-12% SDS-PAGE gels commonly providing optimal resolution for the 48.1 kDa STK25 protein . Primary antibody concentration requires careful titration, typically starting with the manufacturer's recommended dilution (often 1:1000 to 1:2000) and adjusting as needed based on signal-to-noise ratio. Incubation conditions also warrant optimization, with many protocols utilizing overnight incubation at 4°C to maximize specific binding while minimizing background . Appropriate loading controls should be selected based on the experimental context, with housekeeping proteins such as β-actin or GAPDH commonly used for total protein normalization. For investigations focusing on specific cellular compartments, organelle-specific proteins may serve as more appropriate controls, particularly when examining the lipid droplet association of STK25 or its peroxisomal effects . Signal detection methodology should align with the required sensitivity, with chemiluminescence being widely used for standard applications and fluorescence-based detection offering advantages for quantitative analyses.

How can STK25 phosphorylation status be effectively monitored in research settings?

Monitoring STK25 phosphorylation represents a significant challenge in research settings due to the dynamic nature of phosphorylation events and the specificity required for detection. Phospho-specific antibodies targeting key regulatory sites on STK25 are essential tools for these investigations, though commercially available options remain limited compared to total STK25 antibodies . For in vitro studies examining STK25 activation, researchers can employ kinase assays using purified STK25 protein, which allows for the detection of autophosphorylation activity using general phospho-serine/threonine antibodies or radiometric assays . When studying STK25 phosphorylation in cellular contexts, sample preparation requires rapid lysis at cold temperatures with phosphatase inhibitors to preserve the phosphorylation state. Phos-tag SDS-PAGE represents an advanced technique that can enhance the separation of phosphorylated STK25 isoforms by specifically retarding the migration of phosphorylated proteins, thus allowing visualization of multiple phosphorylation states with a single total STK25 antibody . Mass spectrometry-based phosphoproteomic approaches provide the most comprehensive analysis of STK25 phosphorylation sites but require specialized equipment and expertise. For researchers investigating STK25's role in specific signaling pathways, monitoring the phosphorylation status of known STK25 substrates, such as SAV1 at threonine 26, can serve as functional readouts of STK25 kinase activity .

What are the optimal methods for studying STK25 interactions with binding partners using antibody-based techniques?

Investigating STK25 protein-protein interactions requires specialized antibody-based techniques optimized for preserving native protein complexes. Co-immunoprecipitation (Co-IP) represents the most widely used approach, where STK25 antibodies immobilized on protein A/G beads are used to pull down STK25 along with its interacting partners . The choice of lysis conditions is critical for Co-IP success, with non-denaturing buffers containing mild detergents (such as 0.5% NP-40 or 1% Triton X-100) preserving most protein-protein interactions while maintaining antibody recognition of the target. When studying interactions with components of the STRIPAK complex or the Hippo pathway, researchers should include specific protease inhibitors and maintain lysis temperatures at 4°C to prevent complex dissociation . Proximity ligation assays (PLA) offer an alternative approach for visualizing STK25 interactions in situ, requiring pairs of antibodies targeting STK25 and its suspected binding partner from different host species. For investigating dynamic interactions under various cellular conditions, bimolecular fluorescence complementation (BiFC) or fluorescence resonance energy transfer (FRET) can be employed, though these techniques require expression of tagged proteins rather than direct antibody detection. When evaluating specific binding domains, researchers can design experiments using truncated STK25 constructs and perform domain-mapping studies through Co-IP, as demonstrated in studies exploring STK25's interaction with SAV1 in the Hippo pathway .

How can researchers effectively study STK25 localization to lipid droplets using immunostaining techniques?

Investigating STK25's association with lipid droplets presents unique methodological challenges that require specialized immunostaining protocols. Effective fixation is crucial, with researchers finding that a combination of 4% paraformaldehyde fixation followed by careful permeabilization with low concentrations (0.1-0.2%) of Triton X-100 or saponin preserves both lipid droplet structure and STK25 antigenicity . The timing of permeabilization is critical, as excessive exposure can disrupt lipid droplet morphology while insufficient permeabilization prevents antibody access to STK25. When performing double immunofluorescence staining, researchers should select lipid droplet markers that are compatible with STK25 antibodies in terms of host species origin to avoid cross-reactivity. BODIPY 493/503 or LipidTOX dyes represent useful options for lipid droplet visualization that complement antibody-based STK25 detection . Confocal microscopy with high-resolution Z-stack imaging is essential for accurately determining whether STK25 localizes to the lipid droplet surface or interior. When studying STK25 localization dynamics, researchers have found that oleic acid treatment of hepatocytes effectively induces lipid droplet formation and can enhance visualization of STK25 recruitment patterns . For quantitative analyses, automated image analysis tools should be employed to measure colocalization coefficients between STK25 and lipid droplet markers across multiple cells and experimental conditions.

What strategies exist for simultaneously detecting STK25 and assessing peroxisomal function?

The relationship between STK25 and peroxisomal function requires sophisticated experimental approaches that combine STK25 detection with assessments of peroxisomal biology. Dual immunofluorescence staining using STK25 antibodies together with antibodies against peroxisomal markers such as PEX5 (peroxisome biogenesis marker) enables visualization of spatial relationships between STK25 and peroxisomes . For functional studies, researchers have developed protocols that combine siRNA-mediated STK25 knockdown with subsequent analysis of peroxisomal enzyme activities, particularly those involved in fatty acid oxidation pathways . When implementing these protocols, validation of siRNA efficiency using both qPCR and Western blotting with anti-STK25 antibodies is essential for establishing the relationship between STK25 depletion levels and observed peroxisomal phenotypes. Metabolic flux analyses using radiolabeled fatty acid substrates provide quantitative measurements of peroxisomal beta-oxidation rates in the presence or absence of STK25. For detecting peroxisome-generated reactive oxygen species in the context of STK25 manipulation, probes such as dihydroethidium (DHE) have been successfully employed alongside STK25 immunostaining . The use of 4-hydroxynonenal (4-HNE) staining as a marker of oxidative damage can be combined with STK25 detection to assess the relationship between STK25 levels and oxidative stress in various tissues and experimental conditions .

How should researchers approach STK25 antibody selection for cross-species studies?

Cross-species studies of STK25 require careful antibody selection to ensure consistent detection across different model organisms. Sequence alignment analysis reveals significant conservation of STK25 across mammalian species, though specific epitope regions may contain amino acid variations that affect antibody recognition . When planning multi-species studies, researchers should prioritize antibodies raised against highly conserved regions of STK25, typically within the catalytic domain which maintains greater evolutionary conservation than N-terminal or C-terminal regions . Validation across target species is essential, ideally through Western blotting of tissue samples from each species to confirm specificity and sensitivity. Some commercially available antibodies specify cross-reactivity with multiple species (human, mouse, rat, canine, porcine, etc.), though these claims should be independently verified before conducting extensive experiments . For immunohistochemistry applications across species, researchers should optimize fixation and antigen retrieval protocols individually for each species, as tissue composition differences can significantly impact antibody accessibility and background staining. When antibodies with appropriate cross-reactivity cannot be identified, researchers may need to employ species-specific antibodies and ensure that experimental conditions and detection methods are standardized to allow meaningful comparisons of STK25 expression or localization patterns between species.

What considerations are important when studying STK25 in metabolic disease models?

Investigating STK25 in metabolic disease models requires specialized approaches that address the complex interplay between STK25 signaling and metabolic pathways. When selecting antibodies for these studies, researchers should consider the specific metabolic tissues of interest (liver, adipose tissue, skeletal muscle) and validate STK25 antibody performance in each relevant tissue type . Diet-induced obesity models have been particularly informative for STK25 research, with studies demonstrating that STK25 depletion protects against high-fat diet-induced lipid accumulation and inflammation in adipose tissues . When designing such experiments, researchers should incorporate comprehensive metabolic phenotyping alongside STK25 protein analysis, including measurements of tissue triglyceride content, inflammatory markers, and insulin signaling components. The timing of sample collection is critical in metabolic studies, as STK25 expression and activity may vary with feeding/fasting cycles and disease progression. For mechanistic investigations linking STK25 to specific metabolic pathways, researchers have successfully combined STK25 antibody-based detection with analyses of proteins such as FABP1 and GSTT2/GSTT3, which show altered expression levels in STK25-deficient models . When studying STK25 in human metabolic disease, liver biopsies from patients with varying degrees of steatosis or NASH can be analyzed for STK25 expression and localization patterns, though careful standardization of sample collection and processing is essential for meaningful comparisons.

What methods are available for studying STK25's role in the Hippo signaling pathway?

Investigating STK25's function within the Hippo signaling pathway requires specialized experimental approaches focused on protein-protein interactions and phosphorylation cascades. Co-immunoprecipitation studies using STK25 antibodies have successfully demonstrated interactions with key Hippo pathway components, particularly the complex formed between STK25 and SAV1-STRIPAK . When designing these experiments, researchers should carefully optimize lysis conditions to preserve native protein complexes while maintaining antibody specificity. For analyzing the functional impact of STK25 on Hippo pathway activation, monitoring the phosphorylation status of downstream effectors like MST1/2, LATS1, and YAP has proven effective, with STK25 depletion shown to enhance MST2 activation without affecting STRIPAK complex integrity . The generation of STK25 knockout cell lines using CRISPR/Cas9 technology, followed by assessment of Hippo pathway component phosphorylation status using specific phospho-antibodies, represents a powerful approach for mechanistic studies . Researchers investigating the kinase-dependent functions of STK25 should include experiments with kinase-dead mutants (such as STK25 D158N) to distinguish between scaffold and enzymatic functions . For reconstitution experiments, purified STK25 protein can be produced in bacterial expression systems (such as BL21(DE3)-T1R cells) and purified using standard approaches including Ni-NTA affinity chromatography, anion-exchange chromatography, and size-exclusion chromatography .

How can researchers address common challenges with STK25 antibody specificity?

Antibody specificity issues represent a significant challenge in STK25 research, requiring systematic troubleshooting and validation approaches. Cross-reactivity with related GCKIII family kinases (MST3 and MST4) is a particular concern due to sequence homology, necessitating careful selection of antibodies targeting unique regions of STK25 . When encountering multiple bands in Western blot applications, researchers should first verify sample quality and preparation, as proteolytic degradation can generate STK25 fragments. Additional validation steps include performing pre-adsorption tests with immunizing peptides to confirm specificity, and comparative analysis using multiple antibodies targeting different STK25 epitopes. For studies focusing on endogenous STK25, researchers should consider generating new STK25 knockout controls using CRISPR/Cas9 technology if knockout mouse tissues are unavailable, as these provide the most definitive negative controls . An effective approach in cell culture systems involves siRNA-mediated knockdown using multiple independent siRNA sequences (such as siSTK25 #1: GAAGGUGCCCUGUGCUAUGdTdT and siSTK25 #2: CGGAGCAGGGUGACGUGAAdTdT) to demonstrate specific signal reduction correlating with knockdown efficiency . When studying STK25 in complex tissues, it may be necessary to enrich for specific cell populations through techniques such as fluorescence-activated cell sorting or laser capture microdissection prior to Western blotting to enhance detection of cell type-specific expression patterns.

What strategies can improve detection of low-abundance STK25 in certain tissues or subcellular fractions?

Detecting low-abundance STK25 in specific tissues or subcellular compartments presents technical challenges that require specialized approaches. For tissues with low STK25 expression, sample enrichment through subcellular fractionation can concentrate STK25 from specific compartments, such as lipid droplet fractions in hepatocytes or adipocytes . Researchers studying STK25 in lipid-rich tissues should optimize protein extraction protocols, as standard RIPA buffers may not efficiently solubilize lipid-associated proteins; alternative approaches include the use of stronger detergents or specialized lipid droplet isolation procedures followed by Western blotting . Signal amplification strategies for Western blotting include using high-sensitivity chemiluminescent substrates, increasing antibody concentration, or employing biotinylated secondary antibodies with streptavidin-HRP systems. For immunohistochemistry or immunofluorescence applications, tyramide signal amplification (TSA) can significantly enhance detection sensitivity while maintaining specificity. When examining STK25 localization in specific organelles, super-resolution microscopy techniques such as structured illumination microscopy (SIM) or stimulated emission depletion (STED) microscopy can provide improved visualization of STK25 distribution relative to organelle markers. For quantitative analyses of low-abundance STK25, researchers have successfully employed targeted mass spectrometry approaches, such as selected reaction monitoring (SRM) or parallel reaction monitoring (PRM), using synthetic STK25 peptide standards for absolute quantification across different experimental conditions.

How should researchers interpret contradictory findings regarding STK25 localization or function?

Reconciling contradictory findings regarding STK25 localization or function requires careful consideration of methodological differences across studies. Cell type-specific effects represent a common source of apparent contradictions, with STK25 potentially exhibiting different subcellular localizations or functions depending on the cellular context . When facing such discrepancies, researchers should thoroughly compare experimental conditions, including cell types, culture conditions, and stimulation protocols used across different studies. Differences in antibody specificity or epitope accessibility may also contribute to contradictory localization findings, necessitating validation with multiple independent antibodies targeting different regions of STK25. For functional studies yielding conflicting results, the extent of STK25 depletion or overexpression should be quantitatively assessed, as partial knockdown may produce different phenotypes compared to complete knockout. The presence of compensatory mechanisms involving related kinases (MST3 and MST4) may explain different outcomes in acute knockdown versus genetic knockout models, making it advisable to examine the expression of these related kinases when interpreting STK25 functional data . Researchers should also consider potential strain-specific or sex-specific effects in animal models, as genetic background and hormonal factors can significantly influence STK25-related phenotypes, particularly in metabolic disease contexts .