PRKAA2 Antibody, HRP conjugated

What is PRKAA2 and why is it important for metabolic research?

PRKAA2 (Protein Kinase AMP-Activated Catalytic Subunit Alpha 2) is a catalytic subunit of AMP-activated protein kinase (AMPK), functioning as an energy sensor protein kinase that plays a key role in regulating cellular energy metabolism. In response to reduction of intracellular ATP levels, AMPK activates energy-producing pathways and inhibits energy-consuming processes, including protein, carbohydrate, and lipid biosynthesis, as well as cell growth and proliferation .

PRKAA2's significance in research stems from its multiple roles:

• Regulates lipid synthesis by phosphorylating metabolic enzymes (ACACA, ACACB, GYS1, HMGCR, LIPE)

• Controls insulin signaling and glycolysis by phosphorylating IRS1, PFKFB2, PFKFB3

• Stimulates glucose uptake in muscle by increasing SLC2A4/GLUT4 translocation

• Regulates transcription by phosphorylating factors involved in energy metabolism

What applications are HRP-conjugated PRKAA2 antibodies most suitable for?

HRP-conjugated PRKAA2 antibodies are particularly well-suited for:

The specificity of HRP-conjugated antibodies makes them ideal for detecting both total PRKAA2 and specific phosphorylated forms (pThr172, pS345, pS491) depending on the epitope targeted .

How do storage conditions affect the performance of HRP-conjugated PRKAA2 antibodies?

Proper storage is critical for maintaining optimal HRP enzyme activity and antibody binding capacity:

• Short-term storage (up to 1 month): Store at 4°C

• Long-term storage: Store at -20°C in small aliquots to prevent freeze-thaw cycles

• Avoid repeated freeze-thaw cycles which can diminish HRP activity and antibody binding

• Most commercial preparations contain stabilizers (often glycerol at 50%) and preservatives (sodium azide at 0.02-0.09%)

Research has shown that HRP activity can decrease by approximately 20-30% after 5 freeze-thaw cycles, potentially affecting detection sensitivity in critical AMPK pathway analyses .

How can researchers distinguish between PRKAA1 and PRKAA2 signals when using antibodies that may recognize both isoforms?

Distinguishing between PRKAA1 (AMPKα1) and PRKAA2 (AMPKα2) requires careful experimental design:

• Isoform-specific antibodies: Select antibodies that specifically target unique regions of PRKAA2. The calculated molecular weight of PRKAA2 is 62320 Da, which is often observed as a 62-63 kDa band in Western blots .

• Knockout validation: Use PRKAA2 knockout/knockdown samples as negative controls. Multiple studies have employed siRNA targeting PRKAA2 to verify antibody specificity .

• Phosphosite-specific detection: When studying regulation, use antibodies targeting specific phosphorylated residues unique to PRKAA2, such as:

  • Phospho-S345 - critical for PRKAA2-specific regulation

  • Phospho-S491 - differentially regulated compared to corresponding sites on PRKAA1

  • Phospho-Thr172 - shared between isoforms but essential for activation

• Tissue expression patterns: PRKAA2 shows tissue-specific expression different from PRKAA1, with higher expression in skeletal muscle and cardiac tissue, which can help in experimental design .

What are the most effective methods for optimizing signal-to-noise ratio when using HRP-conjugated PRKAA2 antibodies?

Optimizing signal-to-noise ratio is crucial for detecting specific PRKAA2 signals, especially when studying low-abundance phosphorylated forms:

• Blocking optimization:

  • Use 0.5% BSA in TBS for reduced background with phospho-specific antibodies

  • For total PRKAA2 detection, 5% non-fat milk in TBST is often effective

• Antibody concentration titration:

  • Start with manufacturer-recommended dilutions (e.g., 1:500-1:5000 for WB)

  • Perform sequential dilutions to determine optimal concentration

• Enhanced washing protocols:

  • Increase washing stringency with 0.1-0.3% Tween-20 in TBS

  • Extended washing times (5 × 5 minutes) improve signal clarity for phospho-PRKAA2 detection

• Substrate selection:

  • For low abundance PRKAA2 phospho-forms, enhanced chemiluminescence substrates with higher sensitivity are recommended

  • For quantitative analyses, consider using fluorescent secondary antibodies instead of HRP conjugates

How can researchers effectively design time-course experiments to study AMPK activation dynamics using HRP-conjugated phospho-specific antibodies?

AMPK activation is a dynamic process that requires careful experimental design:

• Temporal resolution planning:

  • AMPK Thr172 phosphorylation typically peaks at 5-30 minutes after stimulus

  • S345 and S491 phosphorylation may show different kinetics

  • Include both early (0, 5, 15, 30 min) and late (1, 2, 6, 24 hr) time points

• Stimulus selection:

  • Energy depletion: 2-deoxyglucose, oligomycin

  • Pharmacological activators: AICAR, metformin, EX229 (as used in ZNFX1-mediated autophagy studies)

  • Physiological stimuli: glucose deprivation, hypoxia

• Sample processing optimization:

  • Rapid lysis in phosphatase inhibitor-containing buffers

  • Immediate denaturation in SDS sample buffer for phospho-preservation

  • Consistent protein loading verified by total protein staining methods

• Multi-parameter analysis:

  • Probe for multiple phosphorylation sites (Thr172, S345, S491)

  • Include downstream targets like Acetyl-CoA Carboxylase phosphorylation

  • Monitor changes in subcellular localization via fractionation or immunofluorescence

How should researchers interpret variations in PRKAA2 band patterns in Western blots?

Multiple band patterns for PRKAA2 may have biological significance:

To address variability, researchers should:

• Include proper controls (recombinant PRKAA2, tissue lysates with known expression)

• Use multiple antibodies targeting different epitopes

• Verify with genetic approaches (siRNA, CRISPR/Cas9 knockout)

• Consider phosphatase treatment to confirm phospho-specific signals

What strategies should be employed when validating phospho-specific PRKAA2 antibodies for research applications?

Rigorous validation of phospho-specific PRKAA2 antibodies is essential:

• Phosphatase treatment controls:

  • Treatment of duplicate samples with lambda phosphatase should eliminate signal from phospho-specific antibodies

  • Maintain untreated controls processed identically to confirm specificity

• Pharmacological manipulation:

  • AMPK activators (AICAR, A-769662, metformin) should increase phosphorylation signal

  • Compound C (AMPK inhibitor) should decrease phosphorylation

  • Temporal changes should follow known AMPK activation kinetics

• Genetic validation:

  • Use cells expressing PRKAA2 with phospho-site mutations (T172A, S345A, S491A)

  • Compare signals in PRKAA2 knockout versus wild-type models

  • siRNA knockdown should proportionally reduce signal intensity

• Cross-validation with multiple detection methods:

  • Compare results between Western blot, immunofluorescence, and ELISA

  • Verify subcellular localization of phosphorylated forms using cellular fractionation

  • Consider mass spectrometry-based validation for absolute confirmation

How can researchers address contradictory results when comparing phospho-PRKAA2 levels across different experimental systems?

Contradictory results in PRKAA2 phosphorylation studies often arise from methodological differences:

• Tissue/cell-specific regulation:

  • PRKAA2 regulation varies significantly between tissues (liver, muscle, brain)

  • Different cell types express varying levels of upstream kinases (LKB1, CaMKKβ)

  • Consider tissue-specific AMPK complexes with different β/γ subunit compositions

• Environmental conditions:

  • Nutrient status affects basal AMPK activation

  • Cell confluence can impact AMPK signaling

  • Standardize culture conditions (glucose concentration, serum levels)

• Technical considerations:

  • Lysis buffers affect phospho-epitope preservation

  • Sample processing time impacts phosphorylation status

  • Antibody lot-to-lot variation may occur

• Resolution approaches:

  • Include multiple phosphorylation sites in analysis (Thr172, S345, S491)

  • Verify with functional readouts (downstream target phosphorylation)

  • Employ parallel approaches (activity assays alongside phospho-detection)

How can researchers utilize HRP-conjugated PRKAA2 antibodies to study the role of AMPK in regulating autophagy and mitochondrial dynamics?

PRKAA2 plays important roles in autophagy and mitochondrial dynamics that can be studied using specialized approaches:

• Autophagy studies:

  • Recent research shows PRKAA2 mediates anti-ferroptotic and autophagy functions

  • Co-detection with autophagy markers (LC3-II, p62) using multiplex immunofluorescence

  • Use phospho-ULK1 (Ser555) detection as functional readout of AMPK-mediated autophagy induction

  • Implement flux assays with bafilomycin A1 to assess AMPK's role in autophagosome formation versus degradation

• Mitochondrial dynamics:

  • PRKAA deletion promotes mitochondrial fragmentation in vascular endothelial cells

  • Combine PRKAA2 detection with MitoTracker staining and fission/fusion protein immunolabeling

  • Time-lapse imaging with fluorescently-tagged mitochondria following AMPK manipulation

  • Quantify mitochondrial morphology parameters (length, interconnectivity) in relation to PRKAA2 activation

• Experimental approaches:

  • siRNA-mediated knockdown of PRKAA2 increases mitochondrial fission events (47±4 vs. 26±2 events/cell in 10 min)

  • EX229 (AMPK stimulator) can reverse phenotypes in PRKAA2-deficient cells

  • Combined detection of PRKAA2 with mitochondrial dynamics proteins (DRP1, MFN1/2, OPA1)

What considerations are important when designing experiments to study PRKAA2's role in cancer using HRP-conjugated antibodies?

PRKAA2 has emerged as a potential diagnostic and prognostic marker in cancer research:

• Cancer-specific expression patterns:

  • PRKAA2 expression is significantly upregulated in hepatoblastoma (HB) tissues

  • Prognostic indicators show substantial correlation with PRKAA2 expression

  • Different cancers may show varying PRKAA2 expression and phosphorylation patterns

• Functional studies:

  • PRKAA2 promotes proliferation and inhibits ferroptosis in hepatoblastoma cells

  • Regulates HIF-1α and TFR1 in cancer contexts

  • May serve as both tumor promoter or suppressor depending on cancer type

• Technical approaches:

  • Tissue microarray analysis with HRP-conjugated antibodies

  • Correlation of PRKAA2 expression/phosphorylation with clinical outcomes

  • Combined detection with proliferation markers (Ki-67) and ferroptosis indicators

• Validation in patient samples:

  • Use multiple detection methods (IHC, WB, qRT-PCR)

  • Include proper controls (adjacent normal tissue)

  • Correlate with metabolic parameters (glucose uptake, lipid profiles)

How can multiplex immunoassays be optimized to study PRKAA2 interactions with other signaling pathways?

Studying PRKAA2 in the context of broader signaling networks requires advanced multiplex approaches:

• Sequential immunoblotting:

  • Use mild stripping protocols to preserve membrane integrity

  • Start with phospho-specific antibodies before total protein detection

  • Consider size separation when planning multiplex detection

• Multiplex fluorescence immunohistochemistry:

  • Utilize tyramide signal amplification for sequential HRP-based detection

  • Carefully select fluorophores with minimal spectral overlap

  • Include phospho-PRKAA2 detection alongside pathway components of interest:

    • mTOR pathway (phospho-S6K, phospho-4EBP1)

    • Autophagy (LC3, ULK1)

    • Metabolic enzymes (ACC, HMGCR)

• Proximity ligation assays (PLA):

  • Study direct interactions between PRKAA2 and binding partners

  • Detect activation-dependent interactions with substrates

  • Visualize subcellular compartmentalization of interactions

• Pathway analysis validation:

  • Confirm pathway connections using pharmacological modulators

  • Genetic manipulation of upstream/downstream components

  • Temporal resolution of signaling events following stimulus