PRKAA2 Antibody, Biotin conjugated

What is PRKAA2 and why is it significant in metabolic research?

PRKAA2 represents the catalytic subunit alpha-2 of AMP-activated protein kinase (AMPK), a critical energy sensor protein kinase that plays a fundamental role in regulating cellular energy metabolism. In response to reductions in intracellular ATP levels, AMPK activates energy-producing pathways while inhibiting energy-consuming processes, making it essential in maintaining cellular homeostasis. The protein functions by inhibiting protein, carbohydrate, and lipid biosynthesis, as well as regulating cell growth and proliferation through both direct phosphorylation of metabolic enzymes and long-term effects via transcription regulator phosphorylation . PRKAA2's significance extends to multiple physiological processes including glucose homeostasis, lipid metabolism, insulin signaling, and autophagy regulation, positioning it as a central molecular switch in metabolic research and a potential therapeutic target for metabolic disorders.

What are the key characteristics of biotin-conjugated PRKAA2 antibodies?

Biotin-conjugated PRKAA2 antibodies combine specific PRKAA2 recognition with the versatility of biotin labeling, creating a powerful research tool. These antibodies typically feature several defining characteristics: they are often developed in rabbit hosts with polyclonal properties, ensuring broad epitope recognition . Their biotin conjugation enables strong non-covalent binding with streptavidin/avidin detection systems, facilitating signal amplification for enhanced sensitivity. Most preparations are purified using protein A or protein G, achieving >95% purity . The molecular target weighs approximately 62.32 kDa, and the antibodies show reactivity primarily with human samples, though some variants also recognize mouse, rat, pig, and sheep PRKAA2 . They are typically stored in buffer solutions containing glycerol, preservatives like 0.03% Proclin 300, and maintain stability at -20°C to -80°C for extended periods .

How do phospho-specific PRKAA2 antibodies differ from total PRKAA2 antibodies?

Phospho-specific PRKAA2 antibodies, such as those targeting pThr172 and pThr183, are engineered to selectively recognize PRKAA2 only when phosphorylated at specific regulatory sites . This characteristic fundamentally distinguishes them from total PRKAA2 antibodies which detect the protein regardless of its phosphorylation state. Phospho-specific antibodies are generated using synthetic phosphopeptides as immunogens, typically KLH-conjugated phosphopeptides derived from regions surrounding critical phosphorylation sites . These antibodies enable researchers to monitor AMPK activation status directly, as phosphorylation at Thr172/Thr183 correlates with enzymatic activity. Their applications require special handling considerations, including the mandatory use of phosphatase inhibitors during sample preparation and specific blocking agents (BSA rather than milk proteins) to prevent non-specific interactions. While total PRKAA2 antibodies provide information about expression levels, phospho-specific variants deliver crucial insights into the functional activation state of the kinase, making them indispensable for signaling studies.

What are the optimal conditions for using biotin-conjugated PRKAA2 antibodies in Western blotting?

Western blotting with biotin-conjugated PRKAA2 antibodies requires careful optimization across multiple experimental parameters. Sample preparation should include phosphatase inhibitors when studying phosphorylated forms, with cell lysis performed in RIPA or NP-40 based buffers . When separating proteins, researchers should note that PRKAA2 has a molecular weight of approximately 62.32 kDa, making 10% SDS-PAGE gels optimal for resolution . For phospho-specific detection, blocking should be performed with 5% BSA in TBST rather than milk proteins, which contain phosphatases that could interfere with phospho-epitope detection . Primary antibody dilutions typically range from 1:500 to 1:1000, with overnight incubation at 4°C recommended for optimal binding . Detection should utilize streptavidin-HRP conjugates (typically at 1:5000 to 1:10000 dilution) followed by enhanced chemiluminescence. Common troubleshooting issues include high background (addressed through increased washing frequency) and weak signals (improved by longer exposure times or increased protein loading). Validation should include appropriate positive controls from tissues with known PRKAA2 expression, such as liver or skeletal muscle.

How can researchers optimize immunohistochemistry protocols using biotin-conjugated PRKAA2 antibodies?

Optimizing immunohistochemistry with biotin-conjugated PRKAA2 antibodies requires careful attention to tissue preparation and signal development. For paraffin-embedded sections, researchers should employ 10% neutral buffered formalin fixation followed by heat-induced epitope retrieval in citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) . A critical step unique to biotin-conjugated antibodies is blocking endogenous biotin using commercial avidin/biotin blocking kits to prevent false-positive signals, particularly in biotin-rich tissues like liver, kidney, and brain. Endogenous peroxidase activity should be quenched using 0.3% H₂O₂ in methanol before antibody application. Primary antibody incubation should occur at 4°C overnight at dilutions determined through titration experiments (typically 1:100-1:500) . For signal development, researchers can choose between colorimetric detection using streptavidin-HRP followed by DAB, or fluorescent detection with streptavidin conjugated to fluorophores. Controls must include PRKAA2-positive tissues alongside negative controls using isotype-matched IgG. When studying phosphorylated PRKAA2, rapid tissue fixation is essential to preserve phospho-epitopes, and phosphatase inhibitors should be included in all buffers.

What considerations are important when using biotin-conjugated PRKAA2 antibodies for ELISA applications?

ELISA applications with biotin-conjugated PRKAA2 antibodies require meticulous attention to assay design and quality control. Researchers should begin by selecting high-binding ELISA plates specifically designed for protein adherence. For direct ELISA, antigen coating concentration should be optimized through titration experiments, while sandwich ELISA requires careful selection of capture antibodies targeting different epitopes than the biotin-conjugated detection antibody . Blocking should be performed with 1-5% BSA or casein to minimize non-specific binding. A critical consideration specific to biotin-conjugated antibodies is the prevention of endogenous biotin interference, which can be addressed through sample pre-treatment with streptavidin or through specialized blocking reagents. The working dilution of biotin-conjugated PRKAA2 antibodies typically ranges from 1:500 to 1:2000 and should be determined empirically for each lot . Signal development using streptavidin-HRP followed by appropriate substrate provides sensitive detection, with optimization of development time critical for achieving the desired signal-to-noise ratio. Standard curves using recombinant PRKAA2 protein should be established to enable quantitative analysis, and all samples should be run in triplicate to ensure statistical validity.

How can researchers use biotin-conjugated PRKAA2 antibodies to investigate AMPK pathway activation in cellular stress models?

AMPK pathway activation analysis using biotin-conjugated PRKAA2 antibodies can be performed through multi-parameter experimental approaches. Researchers should design systematic stress induction protocols using established AMPK activators (AICAR, metformin, glucose deprivation) with defined time-course sampling to capture activation dynamics . Western blotting with phospho-specific antibodies (targeting pThr172/pThr183) provides direct evidence of AMPK activation, with phospho-to-total PRKAA2 ratios serving as quantitative activation metrics . The biotin conjugation enables multiplex analysis through streptavidin-based detection systems with different fluorophores, allowing simultaneous assessment of PRKAA2 phosphorylation alongside other stress markers. Researchers should validate pathway specificity using pharmacological inhibitors (Compound C) and genetic approaches (PRKAA2 siRNA/shRNA). Downstream target phosphorylation (ACC, Raptor, ULK1) should be monitored as functional readouts of AMPK activity . For subcellular localization studies, the biotin-streptavidin system can be leveraged for high-sensitivity immunofluorescence microscopy to track stress-induced translocation of phospho-PRKAA2 between cytoplasmic and nuclear compartments. These comprehensive approaches enable researchers to establish mechanistic connections between stress stimuli and AMPK pathway activation with high specificity and temporal resolution.

What strategies can improve detection sensitivity when working with low-abundance PRKAA2 in research samples?

Enhancing detection sensitivity for low-abundance PRKAA2 requires implementing specialized methodological approaches across different applications. For Western blotting, researchers should consider sample enrichment through immunoprecipitation prior to SDS-PAGE separation, taking advantage of the strong biotin-streptavidin interaction for efficient pull-down . Signal amplification can be achieved through tyramide signal amplification (TSA) systems, which can increase sensitivity by 10-100 fold compared to conventional detection methods. When performing immunohistochemistry, antigen retrieval protocols should be carefully optimized, with extended retrieval times (15-20 minutes) in citrate buffer often yielding improved epitope accessibility . For ELISA applications, sandwich ELISA formats typically provide superior sensitivity compared to direct ELISA, with careful selection of matched antibody pairs critical for success. Additional sensitivity enhancements can be achieved through chemiluminescent substrates rather than colorimetric detection, and through extended primary antibody incubation times (overnight at 4°C). When working with clinical samples or primary cells with limited material, researchers should consider proximity ligation assays (PLA) which can detect single protein molecules through rolling circle amplification. Each sensitivity enhancement strategy should be validated with appropriate positive and negative controls to ensure specificity is maintained alongside improved detection limits.

How can researchers integrate PRKAA2 detection with functional AMPK activity assays?

Integrating antibody-based PRKAA2 detection with functional AMPK assays provides comprehensive insights into both enzyme state and activity. Researchers should design parallel workflows that simultaneously measure phospho-PRKAA2 levels via Western blotting or ELISA while assessing AMPK catalytic activity through established functional assays . The gold standard AMPK activity assay involves measuring phosphorylation of the SAMS peptide (a synthetic substrate) in immunoprecipitated AMPK complexes, with results expressed as pmol phosphate incorporated per minute per mg protein. Biotin-conjugated antibodies facilitate efficient immunoprecipitation of AMPK complexes for these activity measurements. For cell-based systems, researchers should monitor phosphorylation of endogenous AMPK substrates (ACC at Ser79, Raptor at Ser792) as proximal functional readouts . The integration should include appropriate controls: AMPK activators (A-769662, compound 991) as positive controls and inhibitors (Compound C) as negative controls. A critical aspect of integration is establishing quantitative correlations between phospho-PRKAA2 signal intensity and functional activity measurements across multiple experimental conditions. This correlation analysis helps determine whether phosphorylation at Thr172/Thr183 directly corresponds to enzymatic activity in specific experimental contexts, or whether additional regulatory mechanisms are involved. Multiparametric data integration using specialized software enables holistic interpretation of AMPK regulation in complex biological systems.

What controls and validation steps are essential when using biotin-conjugated PRKAA2 antibodies?

Implementing rigorous controls and validation is fundamental for generating reliable data with biotin-conjugated PRKAA2 antibodies. Essential positive controls include recombinant PRKAA2 protein for antibody characterization, cell lines with established PRKAA2 expression (HepG2, C2C12), and tissues known to express PRKAA2 (liver, skeletal muscle) . Negative controls should include PRKAA2 knockdown or knockout samples (if available), isotype-matched IgG controls, and peptide competition assays where the antibody is pre-incubated with excess immunizing peptide before application to samples. For phospho-specific antibodies, researchers must include phosphorylation-state controls: samples treated with phosphatase inhibitors versus phosphatase-treated samples, and AMPK activator-treated samples as positive controls . A critical validation step specific to biotin-conjugated antibodies is the assessment of endogenous biotin interference, particularly in biotin-rich tissues, using avidin/biotin blocking kits. Technical specificity validation should include Western blotting to confirm detection at the expected molecular weight (approximately 62 kDa for PRKAA2) . Cross-reactivity with the closely related PRKAA1 isoform should be evaluated, especially when using antibodies targeting conserved regions. For reproducible quantification, standard curves with recombinant protein should be established for ELISA applications, and consistent positive controls should be included across all experimental replicates.

How can researchers troubleshoot non-specific binding issues with biotin-conjugated PRKAA2 antibodies?

Non-specific binding presents a significant challenge when working with biotin-conjugated PRKAA2 antibodies, but systematic troubleshooting approaches can resolve these issues. The most common source of non-specificity with biotin-conjugated antibodies is endogenous biotin interference, especially in biotin-rich tissues like liver, kidney, and brain . This can be addressed by implementing commercial avidin/biotin blocking kits before primary antibody application. For Western blotting applications, unexpected bands may indicate cross-reactivity with related proteins (particularly PRKAA1) or protein degradation products; these can be resolved through more stringent washing conditions (increasing salt concentration to 500mM NaCl) and optimizing antibody dilution (typically testing 1:500 to 1:2000 ranges) . For immunohistochemistry applications, high background staining often results from insufficient blocking; increasing blocking agent concentration (5-10% normal serum) and extending blocking time (1-2 hours) can significantly improve signal-to-noise ratio. When persistent non-specific binding occurs despite these measures, researchers should consider alternative detection systems or pre-absorbing the antibody with tissue/cell lysates from negative control samples. For ELISA applications, non-specific binding to the plate can be reduced by optimizing blocking agents (testing BSA, casein, or commercial blocking buffers) and implementing more thorough washing protocols (increasing wash cycles to 5-7 with longer durations). Each troubleshooting step should be implemented systematically, changing only one variable at a time to identify the specific cause of non-specific binding.

What factors affect the stability and performance of biotin-conjugated PRKAA2 antibodies over time?

Multiple factors influence the stability and performance of biotin-conjugated PRKAA2 antibodies during storage and experimental use. Storage temperature represents a critical variable, with long-term storage at -20°C to -80°C recommended to maintain antibody integrity . Repeated freeze-thaw cycles significantly degrade performance through protein denaturation and biotin conjugate disruption; researchers should aliquot antibodies upon first thaw to minimize this effect. Buffer composition plays an essential role in stability, with preservatives like 0.03% Proclin 300 or 0.02% sodium azide preventing microbial growth, while glycerol (typically 50%) prevents freezing damage at lower temperatures . Exposure to light can cause photobleaching of the biotin moiety, reducing binding efficiency to streptavidin; storing antibodies in amber tubes or wrapped in aluminum foil mitigates this risk. pH fluctuations can affect antibody-antigen binding kinetics, making it important to maintain consistent buffer conditions. For working solutions, protein carriers like BSA (0.5-1%) help prevent antibody adsorption to storage tubes and stabilize dilute solutions . Performance degradation manifests as decreased signal intensity with consistent samples, increased background staining, or altered specificity profiles. Researchers should implement regular quality control testing with standard samples, comparing current results with historical data. For long-term studies, purchasing sufficient antibody from a single lot ensures consistency throughout the project. Proper documentation of storage conditions, handling procedures, and lot numbers facilitates troubleshooting when performance issues arise.

How can biotin-conjugated PRKAA2 antibodies be applied in metabolic disease research models?

Biotin-conjugated PRKAA2 antibodies offer valuable applications in metabolic disease research through their ability to precisely monitor AMPK activation status. In diabetes models, these antibodies enable researchers to track PRKAA2 phosphorylation at Thr172/Thr183 in insulin-responsive tissues, correlating activation patterns with glucose homeostasis parameters . The biotin conjugation facilitates multiplex immunohistochemistry analysis, allowing simultaneous visualization of PRKAA2 activation alongside tissue-specific markers and metabolic stress indicators. For obesity research, researchers can examine PRKAA2 phosphorylation status in various adipose tissue depots, correlating with inflammatory markers and adipokine profiles to understand AMPK's role in adipose tissue dysfunction. In non-alcoholic fatty liver disease studies, biotin-conjugated antibodies enable precise localization of active PRKAA2 within hepatic lobules through high-sensitivity immunohistochemistry, correlating spatial activation patterns with disease progression . The experimental approach should include comprehensive profiling across disease stages, incorporating both phospho-specific and total PRKAA2 detection. For therapeutic development, these antibodies facilitate high-throughput screening of AMPK-activating compounds through ELISA-based phosphorylation assays, with biotin-streptavidin systems providing robust signal-to-noise ratios. Multiparametric analysis integrating PRKAA2 phosphorylation data with metabolomic and transcriptomic profiles can reveal novel insights into AMPK's role in disease pathogenesis and identify potential intervention points.

What emerging applications exist for biotin-conjugated PRKAA2 antibodies in cancer research?

AMPK plays complex roles in cancer biology, functioning as both tumor suppressor and promoter depending on context, making biotin-conjugated PRKAA2 antibodies valuable tools in oncology research. These antibodies enable precise assessment of AMPK activation status in tumor versus normal tissue through immunohistochemistry, with the biotin-streptavidin system providing enhanced sensitivity for detecting subtle activation differences . In tumor metabolism studies, researchers can apply these antibodies to analyze metabolic reprogramming events, correlating PRKAA2 phosphorylation with glycolytic markers and mitochondrial function indicators. The biotin conjugation facilitates sequential multiplexed immunostaining, allowing comprehensive profiling of metabolic signaling networks within the same tissue section. For therapeutic response monitoring, biotin-conjugated antibodies enable high-throughput screening of patient-derived xenografts for AMPK activation following treatment with metabolic-targeting drugs, potentially identifying predictive biomarkers for response . In cancer cell line models, these antibodies can be used in combination with metabolic stress induction (glucose deprivation, hypoxia) to monitor dynamic AMPK activation, correlating with survival markers and resistance mechanisms. Technical considerations for cancer applications include accounting for tumor heterogeneity through careful sampling and incorporating microdissection techniques to isolate specific tumor regions. The experimental design should compare matched tumor-normal pairs when possible and correlate antibody-detected signals with clinical outcomes to establish prognostic relevance of AMPK activation patterns in specific cancer types.

How might biotin-conjugated PRKAA2 antibodies contribute to understanding the role of AMPK in cellular stress and autophagy regulation?

Biotin-conjugated PRKAA2 antibodies provide sophisticated tools for dissecting AMPK's central role in cellular stress responses and autophagy regulation. For stress response profiling, these antibodies enable monitoring of PRKAA2 activation kinetics following various stressors (oxidative stress, ER stress, nutrient deprivation), with the biotin-streptavidin detection system providing the sensitivity needed to detect early activation events . In autophagy pathway analysis, researchers can track AMPK-mediated phosphorylation of critical substrates like ULK1 (Ser317, Ser777) using phospho-specific antibodies, while simultaneously monitoring PRKAA2 activation status . The biotin conjugation facilitates efficient pull-down of AMPK complexes for interaction partner identification, potentially revealing novel stress-specific regulatory proteins. For advanced imaging applications, these antibodies can be combined with super-resolution microscopy to resolve subcellular localization of active AMPK during stress responses, with the biotin-streptavidin system enabling signal amplification for detection of low-abundance complexes. When studying mitochondrial stress responses, biotin-conjugated PRKAA2 antibodies can be used in proximity ligation assays to detect interactions with mitochondrial proteins, revealing mechanisms of AMPK-mediated mitochondrial quality control. The experimental workflow should induce specific stressors, monitor PRKAA2 phosphorylation at defined time points, track downstream events (substrate phosphorylation, autophagic markers), and validate pathway connections using genetic manipulation approaches. Integration of antibody-detected signals with functional autophagic flux measurements provides comprehensive insights into AMPK's role in coordinating cellular adaptation to diverse stress conditions.