PRKACA/PRKACB Antibody
Description
Molecular and Functional Characteristics of PRKACA/PRKACB Antibodies
PRKACA/PRKACB antibodies are designed to detect specific isoforms of PKA catalytic subunits:
Both subunits share a conserved catalytic core but differ in N-terminal hypervariable regions, enabling isoform-specific antibody development . These antibodies are essential for investigating PKA’s roles in cell proliferation, differentiation, and signal transduction .
Table 1: Comparison of PRKACA/PRKACB Antibodies
3.1. Mechanistic Studies
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Disease Modeling: Variants in PRKACA and PRKACB are linked to developmental syndromes involving cardiac defects (e.g., atrioventricular septal defects) and polydactyly . Antibodies help identify hyperactive PKA holoenzymes in these pathologies .
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Cancer Research: PRKACA expression correlates with microsatellite stability in colorectal cancer and chemotherapeutic response in gastric cancer .
3.2. Signaling Pathways
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Hedgehog Inhibition: PRKACB antibodies validated reduced hedgehog signaling in fibroblasts expressing PRKACB mutants .
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Cellular Senescence: PRKACB is implicated in RNA methylation dynamics during fibroblast senescence .
Table 2: Key Findings from PRKACA/PRKACB Antibody-Based Studies
Technical Considerations
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Validation: Proteintech’s 12232-1-AP antibody detects PRKACB at 36–55 kDa (predicted: 41 kDa) , while Bioss’ bs-3725R targets phosphorylated Thr198 .
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Protocols: Optimized dilution ranges vary (e.g., 1:50–200 for IF, 1:500–1000 for ELISA) .
Future Directions
PRKACA/PRKACB antibodies are pivotal for advancing precision oncology and developmental biology. Emerging applications include:
Product Specs
Q&A
How to validate the specificity of PRKACA/PRKACB antibodies in experimental models?
Validation requires a multi-step approach:
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Knockout/knockdown controls: Use cell lines or tissues lacking PRKACA/PRKACB (e.g., CRISPR-edited models) to confirm absence of signal .
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Peptide blocking: Pre-incubate antibodies with immunizing peptides; a >70% reduction in signal indicates specificity .
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Orthogonal validation: Compare results across techniques (e.g., Western blot vs. IHC vs. RNA-seq) . For example, search results demonstrate antibodies like ABIN7184469 (anti-PRKACA N-Term) show consistent 36–55 kDa bands in human/mouse tissues , aligning with PRKACA’s predicted 40.6 kDa mass .
What are the common cross-reactivity issues with PRKACA/PRKACB antibodies, and how are they resolved?
Cross-reactivity arises due to:
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Paralog homology: PRKACA (Cα) and PRKACB (Cβ) share 91% amino acid identity in catalytic domains . Antibodies targeting unique regions (e.g., N-terminal epitopes) reduce overlap .
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Species variability: Anti-PRKACA ABIN7184469 reacts with human/mouse/rat, while PRKACB antibody 55382-1-AP detects primate-specific isoforms .
Mitigation strategies:
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Validate antibodies in species-specific knockout models.
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Use isoform-specific assays (e.g., qRT-PCR) to confirm target expression .
How to optimize PRKACA/PRKACB IHC protocols for FFPE tissues?
Key variables include:
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Antigen retrieval: TE buffer (pH 9.0) outperforms citrate for PRKACB in renal tissues .
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Fixation time: Limit formalin exposure to <24 hrs to prevent epitope masking .
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Validation: Compare staining patterns with Western blot data. For instance, PRKACB antibody 12232-1-AP shows nuclear/cytoplasmic localization in HeLa cells , aligning with PKA’s subcellular roles .
How are PRKACA/PRKACB fusion proteins detected in intraductal oncocytic papillary neoplasms (IOPNs)?
Methodological workflow:
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RNA-based sequencing: Identify fusions (e.g., ATP1B1::PRKACA) via RT-PCR .
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qRT-PCR: Quantify fusion transcript levels (typical IOPNs show 5–10× higher expression vs. atypical) .
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IHC: Use phosphorylation-specific antibodies (e.g., anti-pCREB) to confirm downstream PKA activation .
Key finding: 100% of typical IOPNs harbor PRKACA/B fusions vs. 46% of atypical cases .
How to resolve discrepancies between Western blot and IHC data for PRKACA/PRKACB?
Common causes and solutions:
Example: PRKACB antibody 55382-1-AP detects 36–55 kDa bands in WB , while IHC shows variable staining due to splice variants .
What epitope-specific antibodies are optimal for studying PRKACA/PRKACB post-translational modifications?
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Phosphorylation sites: Anti-pThr197-PRKACA (activates kinase activity) or anti-pSer339-PRKACA (regulates substrate binding) .
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Domain-specific: N-terminal antibodies (e.g., AA 1–30) avoid catalytic domain interference .
Validation: Co-stain with pan-PRKACA/B antibodies to confirm colocalization .
How does tissue heterogeneity impact PRKACA/PRKACB antibody interpretation in tumors?
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Spatial resolution: Single-cell RNA-seq or multiplex IHC distinguishes tumor vs. stromal expression .
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Subclonal analysis: In IOPNs, PRKACA/B fusions localize to oncocytic regions, absent in adjacent normal .
Data example: PRKACA amplifications in adrenal hyperplasias show mosaic expression, requiring microdissection for accurate quantification .
What controls are essential for PRKACA/PRKACB antibody validation in knockout models?
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Negative controls: Knockout tissues + isotype-matched IgG.
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Positive controls: Overexpression systems (e.g., HEK293T transfected with PRKACA/B) .
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Technical controls: Include housekeeping proteins (e.g., GAPDH) for normalization .
How to design experiments studying PRKACA/PRKACB overexpression effects?
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Model selection: Use inducible systems (Tet-On) to avoid constitutive artifacts .
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Phenotypic assays: Measure cAMP response via FRET reporters .
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Pathway analysis: Combine Co-IP (e.g., PRKACA-RIIβ interactions) with phosphoproteomics .
Key finding: PRKACA overexpression inhibits Hedgehog signaling, altering developmental pathways .
