PRKAR2A Antibody, Biotin conjugated

What is PRKAR2A and why is it important in signaling research?

PRKAR2A (also known as PKR2 or PRKAR2) is the cAMP-dependent protein kinase type II-alpha regulatory subunit. It plays a crucial role in cellular signaling as cAMP activates cAMP-dependent protein kinase, which then transduces signals through phosphorylation of various target proteins. The inactive kinase holoenzyme exists as a tetramer composed of two regulatory (like PRKAR2A) and two catalytic subunits. When cAMP binds to the regulatory subunits, it causes dissociation of the inactive holoenzyme, leading to activation of the catalytic subunits and subsequent phosphorylation of downstream targets . PRKAR2A serves as a key regulatory component in numerous physiological processes, making it an important research target across multiple disciplines.

What advantages does biotin conjugation provide for PRKAR2A antibodies?

Biotin conjugation of PRKAR2A antibodies offers several significant methodological advantages:

• Enhanced detection sensitivity: The strong affinity between biotin and streptavidin/avidin (Kd ≈ 10^-15 M) provides highly sensitive detection systems.

• Signal amplification: Multiple streptavidin molecules can bind to a single biotinylated antibody, amplifying detection signals.

• Versatility in detection systems: Compatible with various secondary detection methods including streptavidin-HRP, streptavidin-fluorophores, or streptavidin-gold nanoparticles.

• Reduced background noise: The biotin-streptavidin system typically provides cleaner results with lower non-specific binding compared to conventional secondary antibody approaches .

• Compatibility with complex detection schemes: Particularly valuable in sandwich ELISA systems where the biotin conjugated anti-PRKAR2A antibody serves as the detection antibody .

How is PRKAR2A expression regulated in physiological contexts?

PRKAR2A expression demonstrates context-dependent regulation across various physiological states. In human smooth muscle cells of the uterus, PRKAR2A expression is dynamically regulated during pregnancy. Studies have documented that mRNA and protein expression levels of PRKAR2A substantially increase during pregnancy and decrease upon labor, with changes mirrored by particulate type II PKA activity . This temporal regulation suggests PRKAR2A plays important roles in muscle contractility associated with pregnancy and labor. Additionally, epigenetic mechanisms appear to modulate PRKAR2A gene expression, indicating multiple layers of regulatory control . Understanding these expression patterns is critical for researchers investigating PRKAR2A in reproductive biology or using this system as a model for regulated protein expression.

What detection methods are compatible with biotin conjugated PRKAR2A antibodies?

Biotin conjugated PRKAR2A antibodies are versatile tools compatible with multiple detection platforms:

• ELISA (Enzyme-Linked Immunosorbent Assay): Biotin conjugated anti-PRKAR2A antibodies serve as detection antibodies in sandwich ELISA configurations. After primary antibody binding and washing, the biotinylated detection antibody binds to the target, followed by HRP-Streptavidin addition and subsequent TMB substrate visualization .

• Western Blotting (WB): Multiple validated biotin conjugated PRKAR2A antibodies are available for western blot applications, offering sensitivity for detecting the protein at its observed molecular weight of approximately 40-45 kDa .

• Immunohistochemistry (IHC): Biotin conjugated antibodies can be used for tissue section staining, with signal amplification via streptavidin-HRP systems .

• Immunofluorescence (IF): For cellular localization studies, fluorophore-conjugated streptavidin provides visualization of biotinylated anti-PRKAR2A antibodies .

• Immunoprecipitation (IP): Several biotinylated antibody preparations have been validated for immunoprecipitation applications, allowing protein complex isolation studies .

The selection of detection method should align with research objectives, considering factors like sensitivity requirements, sample type, and available detection instruments.

How should PRKAR2A antibodies be stored and handled to maintain optimal activity?

Proper storage and handling of biotin conjugated PRKAR2A antibodies are essential for maintaining reactivity and specificity:

• Long-term storage: Store at -20°C for up to one year in the recommended buffer (typically containing glycerol as a cryoprotectant) .

• Short-term storage: For frequent use over periods of up to one month, store at 4°C to minimize freeze-thaw cycles .

• Avoid freeze-thaw cycles: Repeated freezing and thawing significantly diminishes antibody activity. Aliquot antibodies before freezing if multiple uses are anticipated .

• Buffer composition: Most commercial preparations contain phosphate buffered saline (pH 7.4), 150mM NaCl, with preservatives like 0.02% sodium azide, and stabilizers such as 50% glycerol and BSA (0.4-0.5mg/ml) .

• Working dilutions: Prepare working dilutions immediately before use and discard unused diluted antibody. Concentrated stock solutions demonstrate better stability than diluted preparations.

• Contamination prevention: Use sterile technique when handling antibody solutions to prevent microbial contamination.

Following these guidelines ensures maximum antibody performance across multiple experimental applications.

What are the optimal dilution factors for different applications of biotin conjugated PRKAR2A antibodies?

Optimal dilution factors vary by application type and specific antibody preparation:

These ranges serve as starting points for optimization. Researchers should perform dilution series experiments to determine the optimal antibody concentration for their specific experimental conditions, sample type, and detection system. For precise recommendations regarding specific commercial antibodies, manufacturers' datasheets should be consulted, as binding characteristics can vary between clones and production lots .

How can phosphorylated forms of PRKAR2A be specifically detected?

Detecting phosphorylated PRKAR2A requires specialized antibodies that recognize specific phosphorylation sites:

• Phospho-specific antibodies: For detection of specific phosphorylation sites like Ser99, specialized phospho-specific antibodies such as Anti-Phospho-PKA R2 (S99) are available . These antibodies are generated using synthetic phosphopeptides corresponding to the residues surrounding the phosphorylation site.

• Validation approaches: Confirm phospho-specificity through:

  • Dephosphorylation controls (e.g., alkaline phosphatase treatment)

  • Comparisons between stimulated vs. unstimulated samples

  • Phosphatase inhibitor treatments

  • Kinase activator/inhibitor treatments

• Sample preparation considerations: Preserve phosphorylation status by:

  • Incorporating phosphatase inhibitors in lysis buffers

  • Maintaining samples at 4°C during processing

  • Using appropriate SDS-PAGE conditions that maintain phosphoepitopes

• Application-specific protocols: For Western blotting, blocking with 5% BSA rather than milk is recommended, as milk contains phosphoproteins that may interfere with phospho-antibody binding .

• Molecular weight considerations: Phosphorylated PRKAR2A typically shows an observed molecular weight of approximately 40 kDa compared to the calculated molecular weight of 45.5 kDa .

When investigating signaling pathways like the JAK2/STAT1 axis, phospho-specific PRKAR2A antibodies can provide critical insights into regulatory mechanisms controlling cellular processes such as neuronal apoptosis .

What controls should be included when working with PRKAR2A antibodies in signaling pathway studies?

Rigorous experimental controls are essential when studying PRKAR2A in signaling contexts:

• Positive controls:

  • Cell lines with confirmed PRKAR2A expression (e.g., those used in antibody validation)

  • Recombinant PRKAR2A protein standards

  • Tissue samples with documented high expression (e.g., uterine smooth muscle during pregnancy)

• Negative controls:

  • PRKAR2A knockout/knockdown cells

  • Isotype-matched control antibodies

  • Primary antibody omission controls

  • Pre-absorption with immunizing peptide

• Pathway modulation controls:

  • cAMP level modulation (e.g., forskolin treatment to increase cAMP)

  • PKA inhibitors (e.g., H-89, PKI)

  • Phosphodiesterase inhibitors to prevent cAMP degradation

  • For JAK2/STAT1 pathway intersection studies: JAK2 inhibitors (e.g., AG490) or activators (e.g., ML228)

• Expression controls:

  • Multiple housekeeping genes/proteins

  • Total protein staining for normalization

  • Loading controls appropriate for subcellular fractions being analyzed

• Antibody validation:

  • Secondary-only controls

  • Testing multiple antibody clones against the same target

  • Cross-validation with non-antibody methods (e.g., mass spectrometry)

Implementing these controls helps distinguish specific from non-specific signals and provides confidence in experimental outcomes, particularly when investigating complex signaling cascades .

How can researchers troubleshoot low signal issues when using biotin conjugated PRKAR2A antibodies?

When encountering weak signals with biotin conjugated PRKAR2A antibodies, consider this systematic troubleshooting approach:

• Sample-related factors:

  • Verify PRKAR2A expression in your samples; expression levels vary significantly between tissues and conditions

  • Consider expression dynamics, as PRKAR2A levels fluctuate with physiological states (e.g., pregnancy)

  • Ensure proper sample preparation with protease and phosphatase inhibitors

  • Optimize protein extraction protocols for membrane-associated proteins

• Technical optimization:

  • Decrease antibody dilution to increase available binding sites

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

  • Enhance signal amplification with multi-step detection systems

  • For ELISA, increase sample concentration and optimize blocking agents

• Detection system enhancements:

  • Verify streptavidin reagent functionality with positive controls

  • Test alternative streptavidin conjugates (HRP vs. fluorophores)

  • Use tyramide signal amplification for immunohistochemistry applications

  • For Western blotting, try more sensitive substrates (e.g., chemiluminescent vs. colorimetric)

• Blocking and washing optimization:

  • Test alternative blocking reagents (BSA vs. casein vs. commercial blockers)

  • Reduce stringency of wash steps (shorter washes, lower detergent concentration)

  • Ensure blocking solution doesn't contain biotin, which would compete with the detection system

• Antibody quality assessment:

  • Verify antibody integrity through dot blot analysis

  • Check for appropriate biotin:antibody conjugation ratio

  • Consider testing antibodies from alternative manufacturers or different clones

If troubleshooting reveals low target expression as the primary issue, consider using more sensitive detection methods like drop-based digital ELISA or proximity ligation assays.

How does PRKAR2A function in the context of neuronal signaling and protection?

PRKAR2A plays significant roles in neuronal physiology and protection through multiple mechanisms:

• Neuronal apoptosis regulation: Research demonstrates that PRKAR2A can repress neuronal apoptosis by inhibiting the JAK2/STAT1 signaling axis. Studies in cerebral ischemia (CI) models show that PRKAR2A overexpression reduces STAT1 phosphorylation levels, thereby attenuating neuronal apoptosis .

• Pathway interactions: PRKAR2A's protective effects involve complex signaling interactions:

  • PRKAR2A overexpression decreases STAT1 phosphorylation

  • JAK2-STAT1 inhibition (using AG490) enhances PRKAR2A's anti-apoptotic effects

  • JAK2-STAT1 activation (using ML228) counteracts PRKAR2A's protective functions

• Circulating biomarker potential: Evidence suggests circ_0000811 functions as a miR-15b sponge that enhances PRKAR2A expression, presenting potential for biomarker development in CI-induced vertigo and other neurological conditions .

• PKA-mediated neuroendocrine functions: PRKAR2A contributes to PKA-related signaling pathways involved in neuroendocrine function through postsynaptic mediation of PKA activity .

• Cyclic nucleotide signaling integration: As a regulatory subunit of PKA, PRKAR2A helps integrate cAMP signaling with other neuronal signaling cascades, affecting processes from neurotransmission to neuroplasticity.

These findings highlight PRKAR2A's neuroprotective potential and suggest it could be a therapeutic target for conditions involving neuronal apoptosis, such as stroke and neurodegenerative diseases .

What methods are optimal for studying PRKAR2A in the context of reproductive biology?

For reproductive biology research involving PRKAR2A, specialized methodological approaches are recommended:

• Tissue-specific considerations:

  • For uterine smooth muscle studies, tissue collection timing is critical as PRKAR2A expression varies significantly with pregnancy stage and labor onset

  • Sample preservation should maintain phosphorylation status for studying PKA activity

  • Consider parallel collection of hormonal data to correlate with PRKAR2A expression

• Expression analysis techniques:

  • Quantitative RT-PCR for mRNA expression dynamics

  • Western blotting with biotin-conjugated antibodies for protein quantification

  • Immunohistochemistry for spatial localization within reproductive tissues

  • PKA activity assays to correlate expression with functional outcomes

• Functional studies:

  • Ex vivo contractility assays in myometrial strips with PRKAR2A modulators

  • cAMP analog treatments to assess regulatory mechanisms

  • siRNA or CRISPR approaches for PRKAR2A knockdown/knockout in reproductive tissue models

  • Correlation of PRKAR2A levels with contractile protein phosphorylation

• Epigenetic regulation assessment:

  • Chromatin immunoprecipitation (ChIP) to study transcription factor binding

  • DNA methylation analysis of the PRKAR2A promoter region

  • Histone modification studies at the PRKAR2A locus

• Translational approaches:

  • Correlation of PRKAR2A expression with clinical parameters in human samples

  • Development of tissue-specific expression systems to model reproductive contexts

  • Integration with hormone receptor signaling pathways relevant to reproductive biology

These methodological approaches enable comprehensive investigation of PRKAR2A's roles in reproductive physiology, particularly in contexts like pregnancy and labor where its expression shows dynamic regulation .

How can PRKAR2A antibodies be used in studying circular RNA regulatory networks?

Recent research has uncovered important roles for PRKAR2A in circular RNA (circRNA) regulatory networks:

• circ_0000811/miR-15b/PRKAR2A axis:

  • Studies have identified that circ_0000811 acts as a miR-15b sponge, indirectly enhancing PRKAR2A expression

  • This regulatory axis has been implicated in neuronal apoptosis in cerebral ischemia-induced vertigo

  • PRKAR2A appears to be a direct target gene of miR-15b, with functional consequences for neuronal survival

• Methodological approaches for studying this network:

  • Dual-luciferase reporter assays to confirm miRNA-target interactions

  • RNA immunoprecipitation to verify circRNA-miRNA-mRNA interactions

  • Biotin-labeled RNA pull-down assays using biotinylated probes against circ_0000811

  • qRT-PCR and Western blotting with biotin-conjugated PRKAR2A antibodies to quantify expression changes

• Analytical considerations:

  • Circular RNA quantification requires specialized primers spanning the back-splice junction

  • Controls should include linear RNA counterparts

  • Bioinformatic prediction tools help identify potential binding sites between circRNAs, miRNAs, and targets

• Experimental validation approaches:

  • Overexpression and knockdown of circ_0000811 to demonstrate effects on PRKAR2A

  • miR-15b mimics or inhibitors to confirm regulatory relationships

  • Rescue experiments (e.g., circ_0000811 overexpression plus miR-15b mimics)

  • Correlation analysis between circ_0000811, miR-15b, and PRKAR2A levels in clinical samples

This emerging research area represents a promising direction for understanding post-transcriptional regulation of PRKAR2A and developing potential therapeutic approaches for neurological conditions involving altered PKA signaling .

What are the most effective methods for studying PRKAR2A phosphorylation dynamics in signaling cascades?

Investigating PRKAR2A phosphorylation dynamics in signaling cascades requires sophisticated methodological approaches:

• Temporal resolution techniques:

  • Time-course experiments with rapid sampling to capture phosphorylation kinetics

  • Pulse-chase approaches using phosphatase inhibitors

  • Live-cell imaging with phospho-specific fluorescent reporters

  • FRET-based biosensors to monitor PKA activity and PRKAR2A conformational changes in real-time

• Site-specific phosphorylation analysis:

  • Phospho-specific antibodies targeting known sites (e.g., S99) for immunoblotting or immunoprecipitation

  • Mass spectrometry-based phosphoproteomics for unbiased site identification

  • Phospho-peptide enrichment techniques (IMAC, TiO2) combined with MS/MS analysis

  • Site-directed mutagenesis (Ser→Ala or Ser→Asp) to create phospho-null or phospho-mimetic mutants

• Signal integration analysis:

  • Multi-pathway inhibitor/activator studies to delineate cross-talk

  • Mathematical modeling of phosphorylation/dephosphorylation kinetics

  • Correlation of PRKAR2A phosphorylation with downstream pathway activation

  • JAK2/STAT1 pathway modulators (AG490, ML228) to explore bidirectional regulation

• Subcellular localization approaches:

  • Fractionation combined with phospho-specific Western blotting

  • Super-resolution microscopy with phospho-specific antibodies

  • Proximity ligation assays to detect interactions with pathway components

  • FRAP (Fluorescence Recovery After Photobleaching) to study phosphorylation-dependent mobility

• Validation strategies:

  • Parallel analysis using multiple detection methods

  • Pharmacological intervention with kinase and phosphatase inhibitors

  • Genetic approaches (CRISPR/Cas9) to introduce phospho-site mutations

  • In vitro kinase assays with purified components

These methodologies enable researchers to characterize the dynamic phosphorylation events that regulate PRKAR2A function in diverse signaling contexts, including its inhibitory role in the JAK2/STAT1 pathway relevant to neuronal apoptosis .