PRKAR2A Antibody, HRP conjugated

What is the specificity profile of PRKAR2A antibody (HRP conjugated)?

PRKAR2A antibody (HRP conjugated) demonstrates high specificity for human PRKAR2A protein, particularly targeting amino acids 43-205 of the regulatory subunit. It is a polyclonal antibody raised in rabbits and purified using Protein G chromatography to achieve >95% purity . This antibody shows minimal cross-reactivity with other protein kinase regulatory subunits, making it suitable for specific detection of PRKAR2A in complex biological samples. When designing experiments, researchers should consider that this antibody has been validated specifically for human samples and may not recognize PRKAR2A from other species with equal efficiency.

What are the primary applications for PRKAR2A antibody (HRP conjugated)?

The primary application for PRKAR2A antibody with HRP conjugation is ELISA (Enzyme-Linked Immunosorbent Assay) . The direct HRP conjugation eliminates the need for secondary antibody incubation steps, simplifying workflow and reducing background. In sandwich ELISA configurations, the antibody functions effectively as a detection antibody when paired with a suitable capture antibody . The enzymatic activity of the HRP conjugate generates a colorimetric readout when exposed to appropriate substrates like TMB (3,3',5,5'-Tetramethylbenzidine), allowing for quantitative assessment of PRKAR2A levels with detection sensitivity in the picogram range.

What is the recommended storage and handling protocol for maintaining antibody activity?

To maintain optimal activity of PRKAR2A antibody (HRP conjugated), the sealed kit should be stored at 2-8°C . After opening, components should be handled according to specific storage requirements, typically involving refrigeration and protection from light exposure to preserve the HRP enzymatic activity. Stability testing indicates that the antibody maintains approximately 95-100% of its activity when stored at 2-8°C for up to 6 months, while storage at elevated temperatures (37°C) for 1 month results in activity retention of approximately 80% . Repeated freeze-thaw cycles should be avoided as they can compromise antibody structure and conjugate stability.

How should I design a sandwich ELISA experiment using PRKAR2A antibody (HRP conjugated)?

When designing a sandwich ELISA for PRKAR2A detection, the microplate should first be coated with a capture anti-PRKAR2A antibody that recognizes a different epitope than the HRP-conjugated detection antibody . After blocking non-specific binding sites, add samples and standards to the wells, followed by incubation with the HRP-conjugated anti-PRKAR2A detection antibody. Post-washing to remove unbound conjugates, add TMB substrate and measure absorbance at 450nm. For optimal results, include a standard curve using recombinant PRKAR2A protein spanning the range of expected concentrations. The assay demonstrates high precision with intra-assay and inter-assay coefficients of variation (CV) around 5%, allowing for reliable quantification of PRKAR2A in serum and plasma samples .

What controls should be included when using PRKAR2A antibody in experimental workflows?

Robust experimental design with PRKAR2A antibody requires several controls. Include negative controls (buffer only or isotype control antibody) to establish background signal levels and non-specific binding. Positive controls using recombinant PRKAR2A protein or well-characterized samples with known PRKAR2A expression provide validation of antibody functionality. For quantitative assays, a standard curve with 6-8 concentration points covering the expected range should be included . When analyzing clinical samples, matrix-matched standards are recommended to account for matrix effects. To verify antibody specificity, consider including samples where PRKAR2A expression has been knocked down or samples from PRKAR2A knockout models like the RIIα-KO mice described in the literature .

How can I determine optimal antibody concentration for my experimental system?

Determining optimal antibody concentration requires titration experiments. Start with the manufacturer-recommended concentration (typically 1-2 μg/ml) and test a range of 2-fold dilutions above and below this value. Evaluate signal-to-noise ratio at each concentration, selecting the dilution that provides robust specific signal while minimizing background. For ELISA applications, construct a standard curve at each antibody concentration and assess parameters like sensitivity, dynamic range, and linearity . The optimal concentration often represents a balance between detection sensitivity and reagent economy. The recovery data from the PRKAR2A ELISA kit indicates good linearity at multiple dilutions (1:2, 1:4, and 1:8), suggesting stable performance across a range of antibody concentrations .

How should I interpret discrepancies between PRKAR2A protein levels and gene expression data?

Discrepancies between PRKAR2A protein levels and gene expression can arise from several factors. Research indicates that PRKAR2A expression is epigenetically regulated through histone deacetylase (HDAC) activity within the GC-rich proximal promoter region . Treatment with HDAC inhibitors like trichostatin A increases both PRKAR2A mRNA and protein levels, indicating regulation at the transcriptional level. When faced with discrepancies, consider: (1) post-transcriptional regulation through microRNAs or circular RNAs derived from the PRKAR2A gene , (2) protein stability or degradation rates, (3) sensitivity differences between protein and mRNA detection methods, and (4) temporal dynamics where protein changes lag behind transcriptional changes. To resolve discrepancies, combine multiple techniques including Western blot, ELISA, qPCR and consider time-course experiments to capture dynamic regulation.

What are common sources of variability in PRKAR2A antibody-based assays and how can they be addressed?

Common sources of variability in PRKAR2A antibody-based assays include sample preparation inconsistencies, antibody lot variations, and assay execution differences. To minimize variability, standardize sample collection and processing protocols, including consistent protein extraction methods and storage conditions. For ELISA applications, precision data indicates typical coefficient of variation (CV) values around 5% . To improve reproducibility: (1) use automated liquid handling where possible, (2) control incubation temperature precisely, (3) prepare fresh reagents according to manufacturer specifications, (4) include internal reference standards on each plate, and (5) perform technical replicates (minimum triplicates). When working with clinical samples, account for matrix effects by using matrix-matched calibrators and performing sample dilution linearity studies as demonstrated in the recovery experiments showing 82-104% recovery across multiple dilutions of serum and plasma samples .

How can I validate that my PRKAR2A antibody is detecting the correct protein isoform?

Validating isoform specificity of PRKAR2A antibody requires multiple approaches. First, review the epitope recognition region of the antibody (amino acids 43-205 for the HRP-conjugated antibody described) and compare with known isoform sequence variations. Second, perform validation experiments using recombinant protein standards of specific isoforms. Third, utilize genetic models with isoform-specific knockdown or knockout, such as the RIIα-KO mice described in the literature . Fourth, compare results with alternative antibodies recognizing different epitopes of PRKAR2A. The catalog information indicates multiple available antibodies targeting different regions of PRKAR2A, including N-terminal (1-105 AA), central region, and full-length (1-404 AA) versions . Cross-validation using these complementary antibodies can confirm isoform specificity. Finally, consider immunoprecipitation followed by mass spectrometry for definitive isoform identification.

How can PRKAR2A antibodies be used to study subcellular localization and compartmentalized signaling?

PRKAR2A antibodies are valuable tools for studying subcellular localization because type II regulatory subunits like RIIα determine the subcellular localization of PKA . For immunofluorescence studies, select unconjugated primary PRKAR2A antibodies compatible with immunofluorescence (IF) applications , followed by fluorophore-conjugated secondary antibodies. Research indicates that PRKAR2A deficiency impairs dendritic localization of PKA catalytic subunits in medial habenula neurons , highlighting the protein's role in compartmentalized signaling. To study dynamic localization, combine with PKA activity sensors or proximity ligation assays. For optimal resolution, consider super-resolution microscopy techniques or electron microscopy using gold-conjugated antibodies. When interpreting localization data, compare with the distribution of A-kinase anchoring proteins (AKAPs), which tether PKA to specific subcellular compartments.

What is the role of PRKAR2A in neuronal function and behavior, and how can it be studied?

PRKAR2A plays critical roles in neuronal function and behavior, particularly through its expression in the medial habenula (MHb) . Studies with RIIα-knockout (RIIα-KO) mice reveal decreased consumption of palatable "rewarding" foods and increased motivation for voluntary exercise . To investigate PRKAR2A's neuronal functions, researchers can: (1) use PRKAR2A antibodies for immunohistochemistry to map expression patterns in specific neuronal populations, (2) combine with markers for neurotransmitter systems, as PRKAR2A is expressed in both substance P and acetylcholine-expressing cells in the MHb , (3) employ electrophysiology to assess how PRKAR2A modulates neuronal activity, and (4) utilize behavioral assays focused on reward processing, feeding behavior, and locomotor activity. The restricted expression of PRKAR2A in glutamatergic neurons but not GABAergic cells suggests pathway-specific functions that can be explored using cell-type-specific manipulations .

How does PRKAR2A contribute to cancer progression, and what experimental approaches can elucidate its role?

Research indicates that PRKAR2A may play significant roles in cancer progression. PRKAR2A-derived circular RNAs promote malignant characteristics in cancer cells , and immunohistochemical studies show that PRKAR2A is primarily expressed on cell membranes in primary colorectal cancer lesions . Overexpression of PRKAR2A correlates with lower 5-year survival rates and higher recurrence in colorectal cancer patients . To investigate PRKAR2A's role in cancer, researchers can: (1) use HRP-conjugated antibodies for immunohistochemical analysis of tumor tissue microarrays, (2) correlate PRKAR2A expression with clinical outcomes, (3) manipulate PRKAR2A expression in cancer cell lines to assess effects on proliferation, migration, and invasion, (4) examine downstream signaling through Western blot analysis of phosphorylation states of STAT3, P65, and β-catenin, which show correlations with PRKAR2A-derived circular RNAs , and (5) employ luciferase reporter assays to study effects on transcriptional regulation, particularly through the Wnt/β-catenin pathway as indicated by TOP/FOP reporter analyses .

How can PRKAR2A antibodies be integrated with high-throughput and single-cell technologies?

PRKAR2A antibodies can be adapted for high-throughput and single-cell applications through several approaches. For high-throughput screening, miniaturized ELISA formats using HRP-conjugated antibodies can assess PRKAR2A levels across large sample sets or treatment conditions . Integration with microfluidic platforms enables reduced sample volumes and increased throughput. For single-cell applications, mass cytometry (CyTOF) using metal-conjugated rather than HRP-conjugated antibodies permits simultaneous measurement of PRKAR2A alongside other proteins. Recent single-cell transcriptomic studies have identified PRKAR2A as a highly and differentially expressed gene among specific cell subsets within the medial habenula , suggesting utility in correlating protein expression with transcriptomic profiles at single-cell resolution. Combining antibody-based detection with laser capture microdissection can isolate PRKAR2A-expressing cells for downstream molecular analysis.

What epigenetic mechanisms regulate PRKAR2A expression, and how can they be experimentally interrogated?

Epigenetic regulation of PRKAR2A occurs through histone modifications within the GC-rich proximal promoter region . Research demonstrates that treatment with the class I/II HDAC inhibitor trichostatin A increases PRKAR2A mRNA and protein levels, preceded by increased acetylated histone H3 (aH3), RNA polymerase II (PolIIa), Sp3, and HDAC2 binding to three SpI-III (GC) binding sites within the PRKAR2A promoter . To investigate these mechanisms, researchers can: (1) perform chromatin immunoprecipitation (ChIP) assays using antibodies against histone modifications and transcription factors, (2) employ luciferase reporter assays with full-length or truncated PRKAR2A promoter constructs to identify regulatory elements , (3) treat cells with epigenetic modifiers like HDAC inhibitors followed by PRKAR2A protein quantification using HRP-conjugated antibodies in ELISA or Western blot, (4) analyze DNA methylation patterns through bisulfite sequencing, and (5) conduct CRISPR-based epigenetic editing to directly manipulate specific regulatory elements.

How do PRKAR2A-derived circular RNAs interact with the protein function, and what methods can investigate this relationship?

PRKAR2A-derived circular RNAs (circRNAs) represent an emerging area of research with implications for both normal physiology and disease states . Studies have identified specific circular RNAs derived from the PRKAR2A gene (including hsa_circ_0124022, hsa_circ_0124028, and hsa_circ_0124029) that may promote malignant characteristics in colorectal cancer . To investigate the relationship between these circRNAs and PRKAR2A protein function, researchers can: (1) quantify both circRNA levels and protein expression using RT-qPCR and ELISA/Western blot with HRP-conjugated antibodies, respectively, (2) perform correlation analyses between circRNA abundance and PRKAR2A protein levels or activity, (3) modulate circRNA levels through overexpression or knockdown approaches and assess consequences on PRKAR2A expression and localization, (4) investigate potential protein-RNA interactions through RNA immunoprecipitation, and (5) examine downstream signaling effects using luciferase reporter assays for pathways like Wnt/β-catenin that show correlation with PRKAR2A-derived circRNAs . The statistically significant correlations observed between these circRNAs and signaling proteins (p-STAT3, p-P65, and β-catenin) suggest functional relationships worthy of detailed mechanistic investigation.