Phospho-PRKAA1/PRKAA2 (Thr183/Thr172) Antibody

What is the biological significance of AMPK phosphorylation at Thr183/Thr172?

The phosphorylation of AMPK at Thr183 (PRKAA1/AMPKα1) and Thr172 (PRKAA2/AMPKα2) represents the activated state of this critical energy sensor. AMPK is a heterotrimeric protein kinase that functions as a cellular energy gauge, responding to changes in the AMP:ATP ratio. When intracellular ATP levels decrease, AMPK becomes phosphorylated at these specific threonine residues, triggering a cascade of events that activate energy-producing pathways while simultaneously inhibiting energy-consuming processes .

This phosphorylation is pivotal for AMPK's ability to:

• Regulate lipid metabolism by phosphorylating and inactivating enzymes like acetyl-CoA carboxylase (ACACA/ACACB)

• Control fatty acid and cholesterol synthesis

• Stimulate glucose uptake by increasing translocation of glucose transporter SLC2A4/GLUT4 to the plasma membrane

• Modulate insulin signaling pathways through phosphorylation of IRS1, PFKFB2, and PFKFB3

• Influence cell growth, proliferation, and polarity

How does the Phospho-PRKAA1/PRKAA2 (Thr183/Thr172) antibody specifically recognize the phosphorylated form?

These antibodies are engineered through a carefully designed immunization and purification process that ensures specificity for the phosphorylated epitope. The process typically involves:

• Immunization of rabbits with synthetic phosphopeptides (sequence around phosphorylation site L-R-T(p)-S-C) conjugated to carrier proteins like KLH

• Affinity purification using epitope-specific phosphopeptide columns

• Negative selection through chromatography with non-phosphopeptides to remove antibodies that might recognize the non-phosphorylated protein

This dual purification strategy ensures the antibody specifically detects endogenous levels of AMPKα1/AMPKα2 only when phosphorylated at Thr183/Thr172, making it an invaluable tool for studying AMPK activation status in various physiological and pathological conditions .

What are the optimal conditions for using Phospho-PRKAA1/PRKAA2 antibodies in Western blotting experiments?

For optimal Western blot results with Phospho-PRKAA1/PRKAA2 (Thr183/Thr172) antibodies, consider the following protocol parameters:

When troubleshooting weak signals, consider:

• Increasing antibody concentration

• Extending incubation time

• Using enhanced chemiluminescence detection systems

• Ensuring samples are fresh and properly preserved with phosphatase inhibitors throughout preparation

How can I verify the specificity of Phospho-PRKAA1/PRKAA2 antibody in my experimental system?

Verifying specificity is critical for phospho-antibodies. Multiple approaches should be employed:

• Phosphatase Treatment Control: Treat a portion of your sample with lambda phosphatase or calf intestinal phosphatase (CIP). The phospho-specific signal should disappear in treated samples while total AMPK signal (detected with a non-phospho-specific antibody) remains unchanged .

• AMPK Activator/Inhibitor Treatment: Treat cells with known AMPK activators (e.g., AICAR, metformin, or glucose deprivation) and inhibitors (e.g., Compound C). The phospho-AMPK signal should increase with activators and decrease with inhibitors.

• siRNA Knockdown: Transfect cells with siRNA targeting PRKAA1/PRKAA2. Both phospho and total AMPK signals should decrease proportionally.

• Peptide Competition Assay: Pre-incubate the antibody with the phospho-peptide immunogen. This should abolish specific binding.

• Molecular Weight Verification: Ensure the detected band appears at the expected molecular weight (~62 kDa) .

These validation approaches help distinguish between specific phospho-AMPK detection and potential cross-reactivity with other phosphorylated proteins.

What are the key differences between polyclonal and monoclonal Phospho-PRKAA1/PRKAA2 antibodies for research applications?

Both polyclonal and recombinant monoclonal Phospho-PRKAA1/PRKAA2 antibodies offer distinct advantages depending on research needs:

For experiments requiring precise quantification across multiple studies over time, recombinant monoclonal antibodies offer superior consistency. For exploratory studies or when detecting proteins in multiple species, polyclonal antibodies may provide advantages due to their broader epitope recognition .

How can I use Phospho-PRKAA1/PRKAA2 antibodies to study AMPK activation in different subcellular compartments?

AMPK can be activated in different subcellular locations, affecting distinct downstream targets. To study compartment-specific activation:

• Immunofluorescence/Confocal Microscopy Approach:

  • Fix cells with 4% paraformaldehyde (10 min, room temperature)

  • Permeabilize with 0.1% Triton X-100 (5 min)

  • Block with 5% BSA in PBS (1 hour)

  • Incubate with Phospho-PRKAA1/PRKAA2 antibody (1:50-1:200 dilution)

  • Co-stain with compartment markers (e.g., DAPI for nucleus, MitoTracker for mitochondria)

  • Use appropriate fluorescent secondary antibodies

• Subcellular Fractionation with Western Blotting:

  • Isolate nuclear, cytoplasmic, mitochondrial, and membrane fractions

  • Verify fraction purity with compartment-specific markers

  • Run Western blots with phospho-AMPK antibodies

  • Compare phospho-AMPK levels across compartments and experimental conditions

• Proximity Ligation Assay (PLA):

  • Use phospho-AMPK antibody together with antibodies against compartment-specific AMPK substrates

  • Detect compartment-specific activation by visualizing interaction signals

These approaches can reveal differential AMPK activation patterns, crucial for understanding the compartmentalized regulation of metabolism and other AMPK-dependent processes.

What are effective strategies for quantifying the ratio of phosphorylated to total AMPK in tissue samples?

Accurate quantification of phospho-to-total AMPK ratios is essential for understanding AMPK activation status. Consider these methodological approaches:

• Sequential Reprobing of Western Blots:

  • First probe with phospho-PRKAA1/PRKAA2 antibody

  • Document results carefully

  • Strip the membrane (validate stripping efficiency)

  • Reprobe with total AMPK antibody

  • Calculate the ratio of phospho/total band intensities using densitometry

• Dual-Color Fluorescent Western Blotting:

  • Use phospho-AMPK and total AMPK antibodies from different species

  • Apply species-specific secondary antibodies with different fluorophores

  • Scan simultaneously to detect both signals

  • Calculate ratios directly without stripping concerns

• ELISA-Based Quantification:

  • Use commercial or self-developed sandwich ELISA

  • Run parallel samples with phospho-AMPK and total AMPK antibodies

  • Generate standard curves for both

  • Calculate the phospho/total ratio

• Mass Spectrometry Approach:

  • Digest samples and enrich for phosphopeptides

  • Quantify phosphorylated and non-phosphorylated peptides containing Thr183/Thr172

  • Calculate stoichiometry based on peptide abundances

For tissue samples specifically, additional considerations include:

• Rapid tissue harvesting and snap-freezing to preserve phosphorylation status

• Homogenization in buffers containing phosphatase inhibitor cocktails

• Normalization to multiple housekeeping proteins for accurate quantification

How should I address potential cross-reactivity with other phosphorylated proteins when using Phospho-PRKAA1/PRKAA2 antibodies?

Cross-reactivity is a significant concern with phospho-specific antibodies. To address this issue:

• Conduct Knockout/Knockdown Validation:

  • Use CRISPR/Cas9 or siRNA to eliminate PRKAA1/PRKAA2 expression

  • Any remaining signal in these samples indicates cross-reactivity

• Phospho-Peptide Competition Assays:

  • Pre-incubate antibody with excess phospho-peptide immunogen

  • Include controls with non-phosphorylated peptide and unrelated phospho-peptides

  • Specific signals should be blocked only by the corresponding phospho-peptide

• Use Multiple Antibodies from Different Sources:

  • Compare results from antibodies raised against different epitopes/regions

  • Consistent results across antibodies increase confidence in specificity

• Employ Phosphatase Treatment Controls:

  • Treat samples with lambda phosphatase

  • Specific phospho-signals should disappear completely

• Bioinformatic Analysis of Similar Phospho-Motifs:

  • Identify proteins with similar phosphorylation motifs to AMPK (L-R-T-S-C)

  • Consider these as potential cross-reactants

  • Verify with specific knockout/inhibition experiments

Manufacturers typically report specificity testing results, but validation in your specific experimental system is critical for reliable data interpretation .

How can I optimize immunohistochemistry protocols with Phospho-PRKAA1/PRKAA2 antibodies for difficult tissue samples?

Optimizing IHC protocols for phospho-AMPK detection in challenging tissues requires careful attention to preservation of phospho-epitopes:

For particularly challenging tissues (brain, muscle, adipose):

• Consider using fresh frozen rather than FFPE samples

• Test multiple antigen retrieval methods (heat vs. enzymatic)

• Include positive controls (tissues from animals treated with AMPK activators)

• Run parallel negative controls (phosphatase-treated sections)

The immunohistochemical images of human lung carcinoma tissue using AMPK-alpha1/AMPK-alpha2(Phospho-Thr174/Thr172) antibody demonstrate the efficacy of properly optimized protocols for detecting phosphorylated AMPK in complex tissue samples .

What are the best experimental approaches for studying AMPK phosphorylation dynamics in response to metabolic stress?

To effectively study dynamic changes in AMPK phosphorylation during metabolic stress:

• Time-Course Experiments:

  • Apply stressor (glucose deprivation, hypoxia, exercise)

  • Collect samples at multiple timepoints (0, 5, 15, 30, 60, 120 min)

  • Analyze phospho-AMPK:total AMPK ratios by Western blot

  • Plot activation kinetics to identify peak activation and resolution

• Live-Cell Imaging with Fluorescent Reporters:

  • Use FRET-based AMPK activity reporters

  • Complement with fixed-cell time points using phospho-antibodies

  • Correlate real-time activity with phosphorylation status

• Metabolic Challenge Protocols:

  • Glucose deprivation (replace with galactose or reduce concentration)

  • Oxygen limitation (hypoxia chambers, 1-5% O₂)

  • ATP synthase inhibition (oligomycin treatment)

  • Exercise mimetics (AICAR, metformin)

  • Monitor phospho-AMPK and downstream targets (ACC phosphorylation)

• Multi-Pathway Analysis:

  • Examine upstream kinases (LKB1, CaMKK2) and phosphatases (PP2A, PP2C)

  • Assess AMP/ATP and ADP/ATP ratios in parallel

  • Correlate AMPK phosphorylation with functional outputs (glucose uptake, lipid oxidation)

Case studies have demonstrated that C2C12 cells show significant AMPK phosphorylation when treated with serum, and this phosphorylation is eliminated by phosphatase treatment, highlighting the importance of both positive and negative controls in such experiments .

How can I distinguish between PRKAA1 (AMPKα1) and PRKAA2 (AMPKα2) phosphorylation in tissue-specific contexts?

While the Phospho-PRKAA1/PRKAA2 (Thr183/Thr172) antibody detects both isoforms due to sequence homology around the phosphorylation site, distinguishing between them is critical for tissue-specific studies:

• Isoform-Specific Immunoprecipitation followed by Phospho-Detection:

  • Immunoprecipitate with isoform-specific antibodies (anti-AMPKα1 or anti-AMPKα2)

  • Probe with phospho-specific antibody

  • Quantify relative phosphorylation of each isoform

• Sequential Immunodepletion:

  • Deplete one isoform with isoform-specific antibodies

  • Analyze remaining phospho-signal attributable to the other isoform

• Utilization of Tissue-Specific Expression Patterns:

  • AMPKα1 predominates in adipose tissue, lungs, and platelets

  • AMPKα2 is more abundant in skeletal muscle, heart, and liver

  • Compare phosphorylation patterns in these tissues

• Genetic Models:

  • Use tissue-specific AMPKα1 or AMPKα2 knockout models

  • Remaining phospho-signal represents the non-deleted isoform

• Mass Spectrometry Approach:

  • Identify and quantify unique phosphopeptides specific to each isoform

  • Calculate the relative abundance of each phosphorylated isoform

Understanding the differential regulation of these isoforms is particularly important in muscle and heart tissues, where AMPKα2 plays a critical role in controlling whole-body insulin sensitivity and maintaining myocardial energy homeostasis during ischemia .

How can Phospho-PRKAA1/PRKAA2 antibodies be utilized in ischemia-reperfusion and metabolic disorder models?

Phospho-PRKAA1/PRKAA2 antibodies are invaluable tools for studying these conditions:

Ischemia-Reperfusion Studies:

• Temporal Analysis:

  • Monitor phospho-AMPK levels during ischemia and following reperfusion

  • Correlate with ATP depletion and recovery

  • Examine differential phosphorylation in affected vs. border zones

• Cardioprotection Research:

  • Compare phospho-AMPK levels in preconditioned vs. non-preconditioned hearts

  • Assess phospho-AMPK in cardioprotective drug treatments

  • Correlate with functional outcomes (infarct size, recovery of function)

Studies suggest that the catalytic subunit PRKAA2 is necessary for maintaining myocardial energy homeostasis during ischemia, making this antibody particularly relevant for cardiovascular research .

Metabolic Disorder Models:

• Insulin Resistance Studies:

  • Assess baseline and stimulated phospho-AMPK in insulin-resistant tissues

  • Compare with healthy controls to identify dysfunctional AMPK signaling

  • Monitor changes during therapeutic interventions

• Peutz-Jeghers Syndrome Research:

  • Examine phospho-AMPK in this LKB1-deficient condition

  • Compare with other related disorders

  • Assess potential therapeutic approaches targeting AMPK activation

• Obesity Models:

  • Measure phospho-AMPK in adipose tissue, liver, and muscle

  • Correlate with metabolic parameters (insulin sensitivity, lipid profiles)

  • Evaluate effects of exercise, caloric restriction, and pharmacological interventions

PRKAA1/PRKAA2 is associated with diseases including Ischemia and Peutz-Jeghers Syndrome, making these antibodies essential for understanding disease mechanisms and developing targeted therapies .

What methodological considerations are important when studying AMPK phosphorylation in neurodegenerative disease models?

Studying AMPK phosphorylation in neurodegeneration requires special considerations:

• Brain Region-Specific Analysis:

  • Different regions show varying AMPK expression and activation patterns

  • Microdissect specific regions before analysis

  • Compare affected vs. unaffected regions within the same sample

• Cell Type-Specific Approaches:

  • Neurons, astrocytes, microglia, and oligodendrocytes have distinct AMPK functions

  • Use dual immunofluorescence with cell-type markers and phospho-AMPK antibodies

  • Consider single-cell analyses for heterogeneous tissues

• Protein Aggregate Considerations:

  • In diseases with protein aggregates (Alzheimer's, Parkinson's), standard extraction may be insufficient

  • Use sequential extraction protocols (detergent-soluble → -insoluble fractions)

  • Examine phospho-AMPK association with disease-specific aggregates

• Post-mortem Tissue Challenges:

  • Phosphorylation status can change rapidly post-mortem

  • Document post-mortem interval and control for this variable

  • Compare results from human samples with fresh animal models

• Specialized Imaging Techniques:

  • For immunohistochemistry, use tyramide signal amplification

  • Recommended antibody dilution: 1:50-1:100

  • Consider super-resolution microscopy for subcellular localization

These approaches help overcome the unique challenges associated with neurodegenerative disease research, where AMPK dysregulation is increasingly recognized as a contributing factor .

How might new developments in phospho-specific antibody technology enhance AMPK research?

Emerging technologies are transforming phospho-antibody capabilities:

• Single-Chain Variable Fragment (scFv) Antibodies:

  • Smaller size allows better tissue penetration

  • Potential for enhanced detection of cryptic phospho-epitopes

  • Development of intrabodies for real-time phosphorylation monitoring

• Phospho-Specific Nanobodies:

  • Single-domain antibody fragments with superior tissue penetration

  • Potential for super-resolution imaging of phospho-AMPK

  • Applications in intravital microscopy for in vivo AMPK dynamics

• Recombinant Antibody Engineering:

  • Creation of isoform-specific phospho-AMPK antibodies through directed evolution

  • Development of antibodies with tunable affinity and specificity

  • Animal origin-free formulations through in vitro expression systems

• Bifunctional Antibody-Based Tools:

  • Proximity-inducing antibodies to study phospho-AMPK interactome

  • PROTAC-conjugated antibodies for selective degradation of phosphorylated targets

  • Optogenetic antibody systems for spatiotemporal control of detection

• Mass Cytometry (CyTOF) Compatible Antibodies:

  • Metal-conjugated phospho-antibodies for simultaneous detection of multiple phosphorylation sites

  • Single-cell resolution of AMPK pathway activation

These emerging technologies will enable researchers to address previously intractable questions about the spatial and temporal dynamics of AMPK activation in complex biological systems.

What are the promising approaches for correlating AMPK phosphorylation with metabolomic and proteomic datasets?

Integrating phospho-AMPK data with -omics approaches offers powerful insights:

• Multi-omics Integration Strategies:

  • Correlate phospho-AMPK levels with global phosphoproteome changes

  • Link AMPK activation to metabolomic shifts in key pathways (glycolysis, TCA cycle, fatty acid metabolism)

  • Create network models connecting AMPK phosphorylation to transcriptional changes

• Temporal Multi-omics:

  • Collect time-series data following AMPK activation

  • Map sequential changes across phosphoproteome → metabolome → transcriptome

  • Identify feedback loops and compensatory mechanisms

• Single-cell Multi-omics:

  • Combine phospho-AMPK immunophenotyping with single-cell RNA-seq

  • Correlate AMPK activation state with cell-specific transcriptional programs

  • Identify heterogeneous responses within tissues

• Spatial Transcriptomics with Phospho-Imaging:

  • Overlay tissue phospho-AMPK immunohistochemistry with spatial transcriptomics

  • Create spatially-resolved maps of AMPK activity and downstream effects

  • Identify microenvironmental factors influencing AMPK activation

• Machine Learning Approaches:

  • Train algorithms to identify metabolic signatures predictive of AMPK activation

  • Develop models relating phospho-AMPK levels to metabolic states

  • Create predictive frameworks for therapeutic interventions targeting AMPK

These integrated approaches will provide systems-level understanding of how AMPK phosphorylation coordinates metabolic adaptation across different physiological and pathological contexts.