Phospho-STK11 (Thr189) Antibody

What is STK11/LKB1 and why is phosphorylation at Thr189 significant?

STK11 (serine/threonine kinase 11), commonly known as LKB1, is a tumor suppressor protein that belongs to the protein kinase superfamily. It functions as a master regulator that controls the activity of AMP-activated protein kinase (AMPK) family members, thereby playing crucial roles in various cellular processes including metabolism, cell polarity, apoptosis, and DNA damage response .

Phosphorylation at threonine 189 (Thr189) represents one of several important post-translational modifications of STK11/LKB1 that can affect its function, localization, and interactions with downstream targets. Unlike some other phosphorylation sites that have been extensively characterized (such as Thr363 and Ser428), the specific regulatory mechanisms and functional consequences of Thr189 phosphorylation are still being investigated by researchers seeking to understand the complete regulatory network of this important tumor suppressor .

What experimental applications are appropriate for Phospho-STK11 (Thr189) antibodies?

Phospho-STK11 (Thr189) antibodies can be utilized in multiple experimental applications for detecting and studying the phosphorylated form of the protein:

These antibodies are particularly valuable for examining changes in STK11/LKB1 phosphorylation status in response to various treatments, signaling events, or disease states . When setting up a new application, it is recommended to perform a titration to determine the optimal working concentration for your specific experimental system.

What is the molecular profile of STK11/LKB1?

STK11/LKB1 has the following molecular characteristics:

The discrepancy between calculated and observed molecular weights is likely due to post-translational modifications such as phosphorylation, which can alter protein migration patterns in SDS-PAGE . When working with this protein, researchers should anticipate detecting bands in the 50-55 kDa range when using Western blot analysis.

How should Phospho-STK11 (Thr189) antibodies be stored and handled?

To maintain antibody integrity and reactivity, proper storage and handling are essential:

• Store at -20°C in aliquots to avoid repeated freeze-thaw cycles

• For conjugated antibodies (e.g., fluorescent-labeled), avoid exposure to light

• Most formulations contain preservatives such as 50% glycerol, 0.5% BSA, and 0.02% sodium azide

• These antibodies typically remain stable for up to one year when stored properly

• Allow the antibody to equilibrate to room temperature before opening the vial

Improper storage or handling can lead to reduced antibody performance, increased background, or false negative results in experimental applications.

How can researchers validate the specificity of Phospho-STK11 (Thr189) antibodies?

Validating antibody specificity is crucial for ensuring reliable experimental results. For Phospho-STK11 (Thr189) antibodies, consider these approaches:

• Phosphatase treatment control: Treat one sample with lambda phosphatase to remove phosphate groups and compare with untreated samples. The signal should decrease or disappear in the treated sample.

• Blocking peptide validation: Use the immunizing phosphopeptide as a competitive inhibitor. The antibody pre-incubated with the phosphopeptide should show significantly reduced or abolished signal compared to the antibody alone .

• Stimulation experiments: Compare samples treated with known modulators of STK11 phosphorylation (e.g., PMA, calyculin A) with untreated controls. Western blot results show enhanced phospho-STK11 (Thr189) signals in NIH/3T3 cells treated with PMA (125ng/ml for 30 minutes) .

• Cross-reactivity testing: Verify that the antibody does not recognize the non-phosphorylated form of the protein or closely related proteins by comparing reactivity patterns.

• Genetic controls: Use STK11 knockout or knockdown models as negative controls to confirm signal specificity.

What cell treatments promote STK11/LKB1 phosphorylation at Thr189?

Several treatments have been shown to modulate STK11/LKB1 phosphorylation status:

Calyculin A is particularly effective as a phosphatase inhibitor that prevents dephosphorylation, thereby maintaining or increasing phosphorylation levels . These treatments provide useful positive controls for validating antibody specificity and studying the regulation of STK11/LKB1 phosphorylation.

How does phosphorylation at different sites affect STK11/LKB1 function?

STK11/LKB1 undergoes multiple phosphorylation events that regulate its activity and interactions:

These phosphorylation events work in concert with other post-translational modifications, such as acetylation at Lys-48, which influences cytoplasmic localization and kinase activity . Understanding the interplay between these modifications is crucial for deciphering STK11/LKB1's complex regulatory network in normal physiology and disease states.

What are the key troubleshooting approaches for Phospho-STK11 (Thr189) antibody applications?

When facing challenges with Phospho-STK11 (Thr189) antibody experiments, consider these methodological solutions:

• Weak or no signal in Western blot:

  • Increase antibody concentration (try 1:500 instead of 1:2000)

  • Use phosphatase inhibitors in lysis buffer to preserve phosphorylation

  • Enrich for phosphorylated proteins using phosphoprotein enrichment kits

  • Treat cells with Calyculin A to increase phosphorylation levels

• High background in immunofluorescence:

  • Optimize blocking conditions (extend blocking time or try different blocking agents)

  • Decrease primary antibody concentration (1:200-1:500 range)

  • Include additional washing steps

  • Use specifically formulated low-background buffers

• Non-specific bands in Western blot:

  • Increase antibody dilution (try 1:5000-1:10000)

  • Optimize blocking conditions

  • Perform peptide competition assay with the phosphopeptide immunogen

  • Run a phosphatase-treated control sample in parallel

• Inconsistent results between experiments:

  • Standardize cell culture conditions and treatments

  • Use positive controls (e.g., Calyculin A-treated cells)

  • Prepare fresh working solutions of antibodies for each experiment

  • Document lot numbers and validate each new lot of antibody

How can Phospho-STK11 (Thr189) antibodies be used to study disease mechanisms?

Phospho-STK11 (Thr189) antibodies are valuable tools for investigating STK11/LKB1's role in various pathological conditions:

• Cancer research:

  • STK11/LKB1 is a known tumor suppressor with defects linked to Peutz-Jeghers syndrome and testicular germ cell tumors

  • Phospho-specific antibodies can help determine if altered phosphorylation states correlate with cancer progression

  • Use multiplex staining with other cancer markers to assess pathway activation in tumor samples

• Metabolic disorders:

  • STK11/LKB1 regulates AMPK, a key metabolic sensor

  • Monitor phosphorylation changes in response to metabolic stress, diabetes models, or obesity

  • Compare phosphorylation patterns between normal and diseased tissues

• Neurodegenerative conditions:

  • STK11/LKB1 plays roles in neuronal polarization and axon specification

  • Investigate phosphorylation status in models of neurodegeneration

  • Correlate with neuronal morphology and function using co-staining approaches

• Developmental studies:

  • Examine phosphorylation patterns during embryonic development

  • Track cell differentiation processes where STK11/LKB1 may play regulatory roles

For these applications, combining phospho-specific antibodies with total STK11/LKB1 antibodies provides a more complete picture of protein regulation and pathway activity.

What are the recommended controls for phospho-specific STK11/LKB1 experiments?

A robust experimental design should include these controls:

• Positive controls:

  • Calyculin A-treated cell lysates (PC-3, HEK-293, or NIH/3T3 cells)

  • PMA-treated NIH/3T3 cells (125ng/ml for 30 minutes)

  • Cells transfected with constitutively active STK11/LKB1

• Negative controls:

  • Untreated cell lysates

  • Phosphatase-treated samples

  • STK11/LKB1 knockout or knockdown samples

  • Secondary antibody-only controls for immunostaining

• Specificity controls:

  • Peptide competition assays using the phosphopeptide immunogen

  • Parallel detection with total STK11/LKB1 antibody

  • Mutation of Thr189 to alanine to prevent phosphorylation

These controls help validate results and troubleshoot any technical issues that may arise during experimentation.

What is the optimal protocol for detecting phosphorylated STK11 at Thr189 by Western blot?

For optimal Western blot detection of phosphorylated STK11 at Thr189:

• Sample preparation:

  • Lyse cells in buffer containing phosphatase inhibitors (e.g., sodium fluoride, sodium orthovanadate)

  • Include protease inhibitors to prevent protein degradation

  • Consider using Calyculin A treatment (50-100 nM for 30 minutes) to enhance phosphorylation signals

• Gel electrophoresis and transfer:

  • Use 10% SDS-PAGE gels for optimal resolution around 50-55 kDa

  • Transfer to PVDF membrane (preferred over nitrocellulose for phosphoproteins)

  • Use wet transfer for more consistent results with phosphoproteins

• Antibody incubation:

  • Block with 5% BSA in TBST (not milk, which contains phosphatases)

  • Dilute primary antibody 1:1000-1:2000 in 5% BSA/TBST

  • Incubate overnight at 4°C with gentle rocking

  • Wash extensively with TBST (4-5 times, 5 minutes each)

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

• Detection:

  • Use enhanced chemiluminescence (ECL) detection systems

  • For weak signals, consider using more sensitive ECL reagents or longer exposure times

  • Expected band size: 50-55 kDa

Following this methodology increases the likelihood of detecting specific phospho-STK11 (Thr189) signals while minimizing background and non-specific binding.

How should researchers quantify changes in STK11/LKB1 Thr189 phosphorylation?

Accurate quantification of phosphorylation changes requires careful normalization and analysis:

• Normalization approaches:

  • Normalize phospho-STK11 signal to total STK11 levels (requires parallel blots or stripping/reprobing)

  • Use housekeeping proteins (e.g., β-actin, GAPDH) as loading controls

  • For immunofluorescence, normalize to total protein staining or cell number

• Quantification methods:

  • For Western blots: Use densitometry software with background subtraction

  • For flow cytometry: Measure mean fluorescence intensity and calculate fold-change

  • For immunofluorescence: Determine integrated density or mean intensity per cell

• Statistical analysis:

  • Perform experiments in triplicate (minimum)

  • Use appropriate statistical tests (t-test for two conditions, ANOVA for multiple conditions)

  • Report results as fold-change relative to control with standard deviation or standard error

• Visualization:

  • Present data as bar graphs with error bars

  • Include representative images of Western blots or immunofluorescence

  • Show both phospho-STK11 and total STK11 signals when available

This systematic approach to quantification ensures reliable and reproducible analysis of phosphorylation changes across experimental conditions.

How can Phospho-STK11 (Thr189) antibodies be used in multi-parameter analyses?

Modern research often requires examining multiple parameters simultaneously:

• Multiplex immunofluorescence:

  • Combine Phospho-STK11 (Thr189) antibodies with antibodies against other signaling molecules

  • Use spectrally distinct fluorophores (e.g., CoraLite® Plus 647 conjugated Phospho-STK11 antibody has excitation/emission maxima at 654/674 nm)

  • Include markers for subcellular compartments to assess localization

  • Analyze co-localization with downstream targets like AMPK

• Phospho-flow cytometry:

  • Utilize conjugated Phospho-STK11 (Thr189) antibodies for intracellular flow cytometry

  • Combine with surface markers to analyze specific cell populations

  • Perform time-course experiments to track phosphorylation dynamics

  • Recommended dilution: 0.06-0.25 μg per 10^6 cells in 100 μl suspension

• Sequential immunoprecipitation:

  • Use Phospho-STK11 (Thr189) antibodies for immunoprecipitation followed by analysis of co-precipitating proteins

  • Identify novel interaction partners specific to the phosphorylated form of STK11/LKB1

  • Compare interactomes between different phosphorylation states

These multi-parameter approaches provide more comprehensive insights into the biological context and functional significance of STK11/LKB1 phosphorylation.

What emerging technologies can enhance Phospho-STK11 (Thr189) research?

Several cutting-edge technologies can advance research using Phospho-STK11 (Thr189) antibodies:

• Mass spectrometry-based validation:

  • Use phospho-enrichment followed by mass spectrometry to validate antibody specificity

  • Quantify absolute levels of phosphorylated vs. non-phosphorylated protein

  • Identify co-occurring post-translational modifications

• Live-cell imaging with phospho-sensors:

  • Develop FRET-based sensors incorporating phospho-specific antibody fragments

  • Monitor real-time changes in STK11/LKB1 phosphorylation in living cells

  • Correlate with cellular functions and responses

• Single-cell analysis:

  • Apply phospho-specific antibodies in single-cell proteomics workflows

  • Capture heterogeneity in phosphorylation status across cell populations

  • Combine with single-cell transcriptomics for multi-omics integration

• In situ proximity ligation assays (PLA):

  • Detect interactions between phosphorylated STK11/LKB1 and binding partners

  • Visualize protein complexes in their native cellular context

  • Quantify interaction frequencies in different subcellular compartments

These technological advances will help researchers gain deeper insights into the dynamic regulation and function of STK11/LKB1 phosphorylation.

What are the key considerations for experimental reproducibility with Phospho-STK11 (Thr189) antibodies?

To ensure reproducible results when working with Phospho-STK11 (Thr189) antibodies:

• Antibody validation and selection:

  • Validate each antibody lot before use in critical experiments

  • Document antibody source, catalog number, and lot number

  • Compare performance across different vendors if possible

• Sample preparation standardization:

  • Standardize cell culture conditions and passage numbers

  • Use consistent lysis buffers with fresh phosphatase inhibitors

  • Process all experimental samples simultaneously

• Technical protocols:

  • Develop detailed standard operating procedures (SOPs)

  • Use consistent antibody dilutions and incubation times

  • Include all appropriate controls in every experiment

• Data analysis and reporting:

  • Use consistent quantification methods

  • Report all experimental details in publications

  • Share raw data when possible to enhance transparency

Following these best practices will enhance the reliability and reproducibility of research findings involving STK11/LKB1 phosphorylation.

How does phosphorylation at Thr189 integrate with other STK11/LKB1 regulatory mechanisms?

STK11/LKB1 function is regulated through a complex network of post-translational modifications and protein interactions:

• Interplay between phosphorylation sites:

  • Phosphorylation at different sites (Thr189, Thr363, Ser428, Ser399) may have cooperative or antagonistic effects

  • Sequential phosphorylation events may create regulatory cascades

  • Different kinases target specific sites under varying cellular conditions

• Integration with other modifications:

  • Acetylation at Lys-48 affects cytoplasmic localization and kinase activity

  • Deacetylation enhances STK11/LKB1 activity

  • Potential cross-talk between phosphorylation and acetylation pathways

• Protein complex formation:

  • STK11/LKB1 functions in complex with STRAD and MO25

  • Phosphorylation may affect complex assembly or stability

  • Different phosphorylation states may recruit distinct binding partners