stk3 Antibody

What is STK3 and why is it important in cellular signaling research?

STK3 (also known as MST2) is a serine/threonine kinase that functions as a key component of the Hippo signaling pathway. This pathway plays a critical role in organ size control and tumor suppression by restricting cellular proliferation and promoting apoptosis. The biological significance of STK3 makes it an important research target because:

• It acts as a stress-activated, pro-apoptotic kinase that, following caspase-cleavage, translocates to the nucleus to induce chromatin condensation and DNA fragmentation

• It forms part of a core kinase cascade wherein STK3/MST2 and STK4/MST1, in complex with regulatory protein SAV1, phosphorylate and activate LATS1/2

• This activation subsequently leads to phosphorylation and inactivation of YAP1 oncoprotein and WWTR1/TAZ, preventing their translocation to the nucleus and regulation of genes involved in cell proliferation, death, and migration

The dysregulation of this pathway has been implicated in various pathological conditions, making STK3 antibodies valuable tools for investigating disease mechanisms and potential therapeutic targets.

Most commercially available STK3 antibodies demonstrate cross-reactivity with multiple species, primarily:

• Human

• Mouse

• Rat

This cross-reactivity is due to the high conservation of STK3 protein sequences across these species. For example, immunogen peptide sequences used in some STK3 antibodies show 94.1% homology between rat and human sequences . When selecting an antibody, researchers should verify the specific species reactivity through:

• Manufacturer validation data

• Published literature using the antibody

• Sequence alignment of the immunogen with the target species' STK3 protein

Note that some antibodies may require additional blocking steps when the host animal matches the target species (e.g., mouse antibodies on mouse tissues) . Researchers should consult technical support for specific protocols in these cases.

How can researchers optimize western blot protocols for detecting STK3 protein?

For optimal western blot detection of STK3 protein, consider these methodological recommendations:

• Sample preparation:

  • Use fresh cell lysates from relevant cell lines (validated cell lines include HeLa, A431, MCF-7, NIH-3T3, C6, Ramos, A20, and L6)

  • Include protease and phosphatase inhibitors in lysis buffers to prevent degradation

  • Denature samples under reducing conditions as STK3 detection is typically performed in reducing environments

• Gel electrophoresis and transfer:

  • STK3 has a molecular weight of approximately 56-65 kDa (observed range)

  • Use PVDF membranes for optimal protein binding and signal detection

• Antibody incubation and detection:

  • Primary antibody dilutions range from 0.04-1 μg/mL depending on the specific antibody

  • For polyclonal antibodies: use HRP-conjugated anti-rabbit/anti-goat secondary antibodies

  • For monoclonal antibodies: use HRP-conjugated anti-mouse secondary antibodies

  • Consider using Immunoblot Buffer Group 1 or 2 as recommended by manufacturers

• Specific technical considerations:

  • STK3 typically appears as a specific band at approximately 56-65 kDa

  • Consider including positive control lysates (A431, HeLa, or MCF-7 cell lines)

  • Full-length STK3 versus caspase-cleaved fragments may appear at different molecular weights, so consider experimental context when interpreting results

What are the best practices for immunofluorescence detection of STK3 in cell culture systems?

To achieve optimal immunofluorescence results when studying STK3 localization:

• Cell preparation and fixation:

  • Validated cell lines include A431 human epithelial carcinoma and NIH/3T3 cells

  • Use immersion fixation (typically 4% paraformaldehyde) to preserve cellular structures

  • Permeabilize cells appropriately (0.1-0.5% Triton X-100) to allow antibody access to intracellular STK3

• Antibody incubation parameters:

  • Primary antibody dilutions typically range from 1:50-1:500 or 0.25-3 μg/mL

  • Incubation times of 1-3 hours at room temperature or overnight at 4°C

  • Use fluorescently-labeled secondary antibodies compatible with your microscope's filter sets (e.g., NorthernLights™ 557-conjugated anti-rabbit IgG)

• Counterstaining and mounting:

  • DAPI nuclear counterstain helps visualize the relationship between STK3 and nuclear structures

  • Use anti-fade mounting medium to preserve fluorescent signal

• Localization patterns to expect:

  • STK3 typically localizes to both cytoplasm and nuclei in many cell types

  • Under apoptotic conditions, expect increased nuclear localization of cleaved STK3

  • Consider co-staining with markers of subcellular compartments to confirm localization patterns

• Controls:

  • Include negative controls (secondary antibody only) to assess background fluorescence

  • Consider siRNA knockdown controls to validate antibody specificity

How should researchers approach STK3 antibody validation to ensure reliable experimental results?

Rigorous antibody validation is essential for generating reproducible and trustworthy research findings. For STK3 antibodies, implement these validation strategies:

• Specificity validation:

  • Genetic approaches: Use STK3 knockout or knockdown (siRNA/shRNA) systems to confirm antibody specificity

  • Peptide competition: Pre-incubate antibody with immunizing peptide to block specific binding

  • Multiple antibody approach: Compare results using different antibodies targeting distinct STK3 epitopes

• Application-specific validation:

  • Western blot: Confirm single band of expected molecular weight (56-65 kDa)

  • IF/IHC: Compare subcellular localization with published patterns (cytoplasmic and nuclear)

  • Flow cytometry: Validate using positive and negative control cell lines

• Documentation of validation experiments:

  • Maintain detailed records of validation procedures

  • Include validation controls in publications

  • Consider requirements of antibody validation initiatives (e.g., Antibodypedia, ENCODE)

• Technical considerations:

  • Batch-to-batch variation: Test new lots against previous results

  • Storage conditions: Follow manufacturer recommendations (typically -20°C, avoid repeated freeze-thaw cycles)

  • Antibody format: Consider if azide-free or BSA-free preparations are needed for specific applications

How can researchers effectively study the role of STK3 in the Hippo signaling pathway using available antibodies?

To investigate STK3's function within the Hippo pathway:

• Phosphorylation status analysis:

  • Use phospho-specific antibodies (when available) to monitor STK3 activation

  • Implement phosphatase treatments as controls to confirm phospho-specificity

  • Design experiments to capture dynamic phosphorylation changes following stimuli like staurosporine or FAS ligand treatment

• Protein-protein interaction studies:

  • Co-immunoprecipitation to study interactions with:

    • Regulatory protein SAV1

    • Downstream targets LATS1/2

    • Other pathway components

  • Proximity ligation assays for in situ visualization of protein interactions

• Subcellular localization dynamics:

  • Live-cell imaging with fluorescently-tagged STK3 to monitor translocation

  • Subcellular fractionation followed by western blot analysis

  • Immunofluorescence under various treatment conditions

• Functional pathway analysis:

  • Monitor downstream targets (LATS1/2, YAP1, TAZ) phosphorylation status

  • Correlate STK3 activity with phenotypic outcomes (proliferation, apoptosis)

  • Design experiments to study feedback regulation within the pathway

• Experimental design considerations:

  • Include positive controls (known pathway activators)

  • Time-course experiments to capture dynamic signaling events

  • Complementary approaches (e.g., genetic manipulation plus antibody-based detection)

What strategies can be used to differentiate between STK3 (MST2) and the closely related STK4 (MST1) in experimental systems?

Distinguishing between the closely related kinases STK3 (MST2) and STK4 (MST1) requires careful experimental design:

• Antibody selection for specificity:

  • Choose antibodies raised against divergent regions between STK3 and STK4

  • Validate antibody specificity using overexpression systems of each kinase

  • Consider using epitope-tagged versions in recombinant systems

• Validation approaches:

  • Western blot: Run STK3 and STK4 recombinant proteins as controls

  • Selective knockdown: Use siRNA targeting only STK3 or STK4 to confirm antibody specificity

  • Mass spectrometry validation of immunoprecipitated proteins

• Functional differentiation strategies:

  • Design experiments leveraging known functional differences

  • Use tissue systems with differential expression patterns

  • Exploit known differential responses to specific stimuli

• Technical considerations:

  • STK3 typically migrates at approximately 56-65 kDa on SDS-PAGE gels

  • Consider possible post-translational modifications that may affect migration patterns

  • Account for tissue-specific expression patterns in experimental design

How can researchers effectively investigate STK3 cleavage and nuclear translocation during apoptosis?

To study the critical process of STK3 cleavage and nuclear translocation during apoptosis:

• Induction of apoptosis:

  • Treat cells with established STK3 activators: staurosporine, FAS ligand

  • Include time-course sampling to capture progression of events

  • Use appropriate apoptosis markers (caspase activation, PARP cleavage) as controls

• Detection of cleaved STK3:

  • Western blot analysis using antibodies that recognize both full-length and cleaved forms

  • Look for appearance of lower molecular weight band corresponding to cleaved STK3

  • Confirm with caspase inhibitors to prevent cleavage as negative control

• Subcellular localization analysis:

  • Subcellular fractionation followed by western blot

  • Immunofluorescence microscopy to visualize nuclear translocation

  • Live-cell imaging with fluorescently-tagged STK3 to monitor real-time dynamics

• Functional assessment:

  • Chromatin condensation assays

  • DNA fragmentation analysis

  • Correlation of nuclear STK3 with apoptotic phenotypes

• Advanced approaches:

  • Generate non-cleavable STK3 mutants to confirm functional significance

  • Implement FRET-based reporters to monitor STK3 cleavage in real-time

  • Use super-resolution microscopy for detailed localization analysis

How can researchers troubleshoot common issues with STK3 antibody performance in experimental applications?

When facing challenges with STK3 antibody applications, consider these troubleshooting approaches:

• Western blot issues:

  • No signal or weak signal:

    • Increase antibody concentration (within recommended range)

    • Optimize protein loading (typically 20-50 μg total protein)

    • Verify transfer efficiency with reversible staining

    • Ensure appropriate secondary antibody compatibility

  • Multiple bands:

    • Increase blocking stringency (5% milk or BSA)

    • Consider longer washing steps

    • Test antibody specificity with blocking peptide

    • Verify if additional bands represent splice variants or degradation products

• Immunofluorescence challenges:

  • High background:

    • Optimize blocking (3-5% BSA or normal serum)

    • Increase washing stringency (longer or additional washes)

    • Decrease primary antibody concentration

    • Include controls for autofluorescence

  • No signal:

    • Verify fixation compatibility (some epitopes are fixation-sensitive)

    • Ensure adequate permeabilization

    • Try antigen retrieval methods

    • Check secondary antibody compatibility

• Flow cytometry optimization:

  • Ensure adequate permeabilization for intracellular staining

  • Titrate antibody to determine optimal concentration

  • Use compensation controls if multiplexing

  • Include appropriate isotype controls

• IHC troubleshooting:

  • Optimize antigen retrieval methods (heat-induced vs. enzymatic)

  • Test different fixatives if possible

  • Vary antibody incubation conditions (time, temperature)

  • Compare chromogenic vs. fluorescent detection systems

What factors should researchers consider when selecting between monoclonal and polyclonal STK3 antibodies for specific applications?

The choice between monoclonal and polyclonal STK3 antibodies depends on experimental goals and technical considerations:

When making this selection, consider:

• Application requirements:

  • Western blot: Both types work well; polyclonals may offer higher sensitivity

  • IHC/IF: Polyclonals may provide better signal in fixed tissues

  • Flow cytometry: Monoclonals often preferred for consistency

  • IP: Consider using the same antibody for IP and detection

• Experimental design factors:

  • Need for consistent supply over long-term projects (favors monoclonals)

  • Detection of denatured vs. native protein

  • Requirement for detecting specific post-translational modifications

  • Multiple species cross-reactivity needs

• Technical considerations:

  • Host species compatibility with experimental system

  • Isotype selection for secondary detection systems

  • Need for direct conjugation to reporters

How can researchers optimize STK3 antibody-based assays to study low abundance expression or challenging tissue samples?

For detecting low-abundance STK3 or working with difficult samples:

• Signal amplification strategies:

  • Implement tyramide signal amplification for IHC/IF

  • Use high-sensitivity chemiluminescent substrates for western blot

  • Consider biotin-streptavidin amplification systems

  • Extend exposure times (while monitoring background)

• Sample enrichment approaches:

  • Immunoprecipitation before western blot

  • Subcellular fractionation to concentrate STK3

  • Optimize protein extraction buffers for target tissue

  • Consider cell sorting to isolate specific populations

• Tissue-specific optimization:

  • Modify fixation protocols (duration, fixative composition)

  • Optimize antigen retrieval methods:

    • Heat-induced epitope retrieval (citrate, EDTA, or Tris buffers)

    • Enzymatic retrieval (proteinase K, trypsin)

  • Implement tissue-specific blocking reagents

  • Extend primary antibody incubation (overnight at 4°C)

• Advanced detection systems:

  • Consider fluorescent western blot for higher sensitivity and quantitative analysis

  • Utilize quantum dot-conjugated secondary antibodies for photostable signal

  • Implement proximity ligation assay for protein interactions

  • Consider multiplex IF with spectral unmixing for complex samples

• Controls and validation:

  • Include positive control samples with known STK3 expression

  • Implement multiple antibody approach (different clones/sources)

  • Consider parallel analysis with mRNA detection methods

How can STK3 antibodies be utilized in studying the role of the Hippo pathway in cancer and therapeutic development?

STK3 antibodies enable multiple approaches to investigate Hippo pathway dysregulation in cancer:

• Expression and activation profiling:

  • IHC analysis of STK3 expression across tumor types and stages

  • Correlation of STK3 expression/phosphorylation with clinical outcomes

  • Analysis of downstream effectors (YAP/TAZ nuclear localization)

  • Comparison between tumor and adjacent normal tissues

• Pathway integrity assessment:

  • Evaluate STK3-LATS-YAP signaling axis in patient samples

  • Correlate pathway disruption with tumor characteristics

  • Study feedback mechanisms and compensatory pathways

  • Assess relation to other oncogenic signaling networks

• Therapeutic response monitoring:

  • Measure STK3 activation following drug treatments

  • Identify biomarkers of response to Hippo pathway-targeted therapies

  • Develop antibody-based assays for patient stratification

  • Monitor pathway reactivation in resistance mechanisms

• Advanced research applications:

  • Proximity-based assays to study STK3 protein interactions in tumor samples

  • Multiplexed IF to analyze pathway components within tumor microenvironment

  • Single-cell analysis of pathway heterogeneity within tumors

  • Extracellular vesicle analysis for circulating biomarkers

What considerations are important when using STK3 antibodies for multiplexed analysis of the Hippo signaling network?

For comprehensive multiplexed analysis of the Hippo pathway:

• Antibody compatibility planning:

  • Select STK3 antibodies raised in different host species than other target antibodies

  • Verify absence of cross-reactivity between antibodies

  • Test for signal bleed-through in multiplexed fluorescence applications

  • Consider sequential staining approaches for challenging combinations

• Multi-parameter experimental design:

  • Include core pathway components: STK3, SAV1, LATS1/2, MOB1, YAP/TAZ

  • Add context-specific markers (cell type, activation state)

  • Plan for appropriate controls for each marker

  • Design panel to answer specific biological questions

• Technical optimization for multiplexing:

  • Titrate each antibody individually before combining

  • Test different orders of antibody application

  • Optimize blocking between sequential staining steps

  • Consider spectral imaging for separating overlapping fluorophores

• Analysis approaches:

  • Implement colocalization analysis for protein interactions

  • Quantify nuclear/cytoplasmic ratios for YAP/TAZ translocation

  • Develop scripts for automated analysis of multiplexed images

  • Consider machine learning approaches for complex pattern recognition

• Emerging technologies:

  • Imaging mass cytometry for highly multiplexed tissue analysis

  • Cyclic immunofluorescence for sequential antibody staining

  • CODEX multiplexed imaging for spatial context

  • Single-cell western blot for heterogeneity analysis

How can researchers effectively use STK3 antibodies in conjunction with phospho-specific antibodies to study pathway activation dynamics?

To comprehensively analyze STK3 activation and signaling dynamics:

• Coordinated antibody selection:

  • Total STK3 antibody: Recognizes protein regardless of phosphorylation state

  • Phospho-STK3 specific antibodies: Target key regulatory phosphorylation sites

  • Downstream substrate antibodies: Monitor pathway activity (phospho-LATS, phospho-YAP)

  • Upstream regulator antibodies: Assess pathway inputs

• Experimental design for activation dynamics:

  • Time-course experiments following stimulus application

  • Parallel sample processing for total and phospho-detection

  • Include phosphatase inhibitors in lysis buffers

  • Prepare activation-state positive controls

• Technical approaches:

  • Western blot: Strip and reprobe membranes for multiple antibodies

  • IF/IHC: Perform sequential or simultaneous staining if antibody species permit

  • Flow cytometry: Use compatible fluorophores for multi-parameter analysis

  • Protein array approaches for higher throughput

• Quantification strategies:

  • Calculate phospho-to-total ratios for normalization

  • Implement kinetic modeling of phosphorylation cascades

  • Correlate STK3 phosphorylation with substrate phosphorylation

  • Consider single-cell approaches to capture population heterogeneity

• Controls and validation:

  • Phosphatase treatment controls

  • Kinase inhibitor controls

  • Genetic manipulation (constitutively active/inactive mutants)

  • In vitro kinase assays for direct activity measurement