PRKAR2A Antibody, FITC conjugated

What is PRKAR2A and what cellular functions does it regulate?

PRKAR2A is the regulatory subunit of cAMP-dependent protein kinase (PKA), a heterotetrameric protein complex consisting of two regulatory and two catalytic subunits. It plays a critical role in modulating the effects of cAMP in various tissues and cell types. PRKAR2A functions as a key component in signal transduction pathways, helping to regulate cellular responses to hormones and other extracellular signals through the cAMP signaling cascade . The protein has a calculated molecular weight of approximately 43-46 kDa but is typically observed between 46-52 kDa in experimental conditions .

What is the difference between FITC-conjugated and unconjugated PRKAR2A antibodies?

FITC-conjugated PRKAR2A antibodies have fluorescein isothiocyanate (FITC) directly attached to the antibody molecule, enabling direct visualization in fluorescence-based applications without requiring secondary antibodies. This makes them particularly valuable for flow cytometry, direct immunofluorescence microscopy, and multiplexed imaging where multiple targets need to be visualized simultaneously . Unconjugated antibodies require additional steps with labeled secondary antibodies for detection but often provide greater flexibility in experimental design and potentially higher sensitivity through signal amplification .

How does phosphorylation at specific residues affect PRKAR2A function in the PKA signaling pathway?

While the search results primarily focus on PRKAR1A phosphorylation rather than PRKAR2A, we can draw some parallels for consideration in research designs. Phosphorylation of regulatory PKA subunits appears to play important roles in modulating PKA activity. For instance, phosphorylation at S77 and S83 on PRKAR1A has been identified in large-scale phosphoproteomic experiments and may influence PKA activity . For PRKAR2A research, it would be valuable to investigate whether similar phosphorylation events occur and how they might regulate protein function. When designing experiments to study PRKAR2A phosphorylation, researchers should consider using phospho-specific antibodies along with standard PRKAR2A antibodies to correlate phosphorylation states with functional outcomes in signaling cascades .

What are the optimal approaches for studying PRKAR2A interactions with other signaling proteins?

Co-immunoprecipitation (IP) experiments are particularly useful for investigating protein-protein interactions involving PRKAR2A. Based on the search results, IP protocols using 0.5-4.0 μg of antibody for 1.0-3.0 mg of total protein lysate have been successfully employed . When studying potential interactions between PRKAR2A and other signaling components, researchers should consider both basal conditions and stimulated states (e.g., forskolin treatment to increase cAMP levels), as these may differentially affect protein interactions . Additionally, combining IP with Western blotting for detection and quantification of interaction partners provides robust data on protein complexes. For advanced studies, consider integrating mass spectrometry analysis of immunoprecipitated complexes to identify novel interaction partners .

How can PRKAR2A antibodies be used to investigate subcellular localization changes in response to signaling events?

FITC-conjugated PRKAR2A antibodies are particularly valuable for tracking subcellular localization through immunofluorescence microscopy. For optimal results in immunofluorescence applications, a dilution range of 1:50-1:500 is typically recommended . When designing experiments to track PRKAR2A translocation during signaling events, consider using co-staining with organelle markers (such as GM130 for Golgi apparatus as mentioned in search result ) to precisely map localization changes. Time-course experiments following stimulation with cAMP-elevating agents can provide insights into the dynamics of PRKAR2A redistribution. For quantitative assessment, confocal microscopy with z-stack imaging followed by colocalization analysis with appropriate software tools can generate robust data on compartment-specific localization changes .

What are the recommended protocols for using FITC-conjugated PRKAR2A antibodies in flow cytometry?

For flow cytometry applications using FITC-conjugated PRKAR2A antibodies, researchers should use approximately 0.40 μg of antibody per 10^6 cells in a 100 μl cell suspension . The protocol should include proper fixation and permeabilization steps since PRKAR2A is primarily an intracellular target. A recommended approach includes:

• Harvest cells and wash twice with PBS

• Fix cells with 4% paraformaldehyde for 10-15 minutes at room temperature

• Permeabilize with 0.1-0.5% Triton X-100 or a commercial permeabilization buffer

• Block with 1-5% BSA or serum for 30 minutes

• Incubate with the FITC-conjugated PRKAR2A antibody at the recommended concentration

• Wash thoroughly to remove unbound antibody

• Analyze by flow cytometry, setting appropriate gates based on negative controls

This method has been validated with cell lines such as MCF-7, but researchers should optimize conditions for their specific cell types .

What are the optimal storage and handling conditions for maintaining PRKAR2A antibody activity?

To maintain PRKAR2A antibody activity, proper storage and handling are crucial. Based on the search results, FITC-conjugated antibodies should be stored at -20°C to -80°C and protected from light to prevent photobleaching of the fluorophore . For long-term storage, aliquoting is recommended to avoid repeated freeze-thaw cycles, although some formulations may be stable without aliquoting for -20°C storage . Most PRKAR2A antibodies are supplied in buffers containing glycerol (typically 40-50%) and preservatives such as sodium azide (0.02-0.05%) or Proclin 300 (0.03%) . When working with these antibodies, allow them to equilibrate to room temperature before opening to prevent condensation, which can degrade protein quality. After use, return to appropriate storage conditions promptly, and handle under reduced light conditions to preserve FITC fluorescence .

What dilution ranges are optimal for different applications of PRKAR2A antibodies?

Different applications require specific antibody dilutions for optimal results. Based on the search results, the following ranges are recommended:

It's important to note that these ranges should serve as starting points, and researchers should perform titration experiments to determine optimal concentrations for their specific samples and detection systems .

How can researchers address discrepancies between expected and observed molecular weights of PRKAR2A in Western blots?

PRKAR2A has a calculated molecular weight of approximately 43-46 kDa but is typically observed between 46-52 kDa in Western blot applications . This discrepancy can be attributed to post-translational modifications, particularly phosphorylation events that alter protein migration. To address this issue, researchers should:

• Always include positive controls with known PRKAR2A expression (e.g., HeLa cells, K-562 cells, or MCF-7 cells as mentioned in search result )

• Consider using phosphatase treatment of some samples to determine if post-translational modifications contribute to the molecular weight shift

• Validate antibody specificity using knockout or knockdown approaches

• Use gradient gels (e.g., 4-12%) to better resolve proteins in this molecular weight range

• Optimize sample preparation methods to minimize proteolysis or other modifications during extraction

Understanding that the observed molecular weight may vary from the calculated weight is important for accurate data interpretation .

What controls are essential when validating PRKAR2A antibody specificity for research applications?

Proper controls are critical for validating PRKAR2A antibody specificity:

• Positive controls: Include samples with known PRKAR2A expression. Cell lines such as HeLa, K-562, MCF-7, and PC-3 have been documented to express detectable levels of PRKAR2A .

• Negative controls:

  • Omission of primary antibody to control for non-specific binding of secondary reagents

  • Use of isotype controls for flow cytometry applications

  • Ideally, PRKAR2A knockout or knockdown samples should be used as gold-standard negative controls

• Peptide competition assays: Pre-incubation of the antibody with the immunizing peptide should abolish specific staining

• Cross-reactivity assessment: If working with multiple species, verify specificity in each relevant model organism

• Multiple detection methods: Confirm findings using alternative techniques (e.g., validate IF results with Western blot data)

These controls help ensure that observed signals are specific to PRKAR2A rather than artifacts or cross-reactivity with other proteins .

How should researchers interpret PRKAR2A localization patterns in different cell types and experimental conditions?

Interpretation of PRKAR2A localization requires careful consideration of cell type, experimental conditions, and proper controls. PRKAR2A typically shows a combination of cytoplasmic and perinuclear localization patterns in resting cells, but this can change dynamically during signaling events. When analyzing localization data:

• Always include suitable subcellular markers to definitively identify compartments (e.g., nuclear, Golgi, endoplasmic reticulum markers)

• Consider that different fixation methods can affect apparent localization patterns; paraformaldehyde fixation is often preferred for maintaining cellular architecture

• Interpret changes in localization in the context of relevant signaling pathways - for example, cAMP elevation often triggers redistribution of PKA regulatory subunits

• Quantify localization patterns across multiple cells and experiments to account for cell-to-cell variability

• Be aware that overexpression systems may produce artifacts compared to endogenous protein localization

• When possible, confirm key findings using super-resolution microscopy techniques for more precise localization data

The search results indicate successful immunofluorescence detection in various samples including mouse testis tissue and cell lines like HeLa, suggesting that PRKAR2A antibodies can effectively visualize the protein in different biological contexts .