PKARIIA (Recombinant)
- Expression system
- HEK293
- Cat. #
- REC-PKARIIA
In stock
- SKU
- REC-PKARIIA
Target Overview
PKARIIA (REC-PKARIIA) is the recombinant human cAMP-dependent protein kinase type IIα regulatory subunit, encoded by PRKAR2A (UniProt P12367, 401 amino acids), produced by transient expression in HEK293 cells. HEK293 expression provides mammalian post-translational processing — including relevant phosphorylation and folding — that is important for preserving the native cAMP-binding and protein–protein interaction surfaces characteristic of this regulatory subunit. In the PKA holoenzyme, the type IIα regulatory subunit (RIIα) forms a tetrameric complex with two catalytic subunits, maintaining the kinase in an inactive state in the absence of cAMP. Upon cAMP elevation, cooperative binding to the two tandem cyclic-nucleotide-binding domains of RIIα induces conformational release of the catalytic subunits. Unlike type I regulatory subunits, type IIα chains are predominantly membrane-associated through direct interaction with A-kinase anchoring proteins (AKAPs), localising PKA activity to discrete subcellular compartments such as the post-synaptic density, mitochondrial outer membrane, and centrosome. Researchers use this recombinant for several in vitro applications: reconstitution of the PKA type IIα holoenzyme for kinase activity and cAMP-dose–response measurements; competitive cAMP-binding assays; AKAP interaction studies using surface plasmon resonance or pull-down formats; and inhibitor/disruptor screens targeting the AKAP–RIIα interface. The full-length, HEK293-expressed format is also a reliable positive-control antigen for antibody validation by Western blot and immunohistochemistry. Researchers validating anti-PRKAR2A antibodies can pair this recombinant with the matched Triple Point Biologics antibody (RP-PKARIIA) to confirm band specificity and immunoreactivity in their experimental system.
Background
Applications
- PKA type IIα holoenzyme reconstitution and catalytic subunit release assay measured by substrate peptide phosphorylation
- Competitive cAMP-binding assay (e.g., fluorescence polarisation or radioligand displacement) to measure RIIα occupancy
- AKAP–RIIα protein–protein interaction studies by surface plasmon resonance or biolayer interferometry
- Small-molecule and peptide inhibitor screen targeting the AKAP–RIIα D/D domain interface
- Western blot positive-control antigen and quantitative standard curve for anti-PRKAR2A detection (pairs with TP antibody RP-PKARIIA)
- Immunohistochemistry antibody validation: spike-in recombinant antigen block to confirm band/signal specificity
- Differential scanning fluorimetry (DSF) thermal shift assay to characterise ligand or cAMP-analogue binding to RIIα
- Pull-down or co-immunoprecipitation substrate for mapping novel AKAP or regulatory interaction partners
References
- Wen J et al. HHEX-PRKAR2B axis-mediated PKA activation drives glucose metabolism-dependent progression of pancreatic ductal adenocarcinoma. iScience. 2026. doi:10.1016/j.isci.2026.114691. PMID: 41704777.
- Gong X et al. Glioma angiogenesis phosphoproteome landscape and biomarker sets identified with phenome-centered multiomics toward 3P medical approaches. EPMA J. 2025. doi:10.1007/s13167-025-00428-1. PMID: 41312000.
- Weng X et al. Analysis of diagnostic apoptosis-related biomarkers and immune cell infiltration characteristics in endometriosis by integrating bioinformatics and machine learning. Sci Rep. 2025. doi:10.1038/s41598-025-19007-5. PMID: 41023129.
- Wang Y et al. Postsynaptic competition between calcineurin and PKA regulates mammalian sleep-wake cycles. Nature. 2024. doi:10.1038/s41586-024-08132-2. PMID: 39506111.
- Mo Y et al. Development and Validation of a Predictive Model for Resistance to Platinum-Based Chemotherapy in Patients with Ovarian Cancer through Proteomic Analysis. J Proteome Res. 2024. doi:10.1021/acs.jproteome.4c00558. PMID: 39253780.
Additional Specifications
| Storage Buffer | 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol |
|---|---|
| Endotoxin Level | <0.1 EU/µg by LAL |
| Purity (%) | >90% by SDS-PAGE |
| Expression System | HEK293 |
| Subcellular Localization | Subcellular localization not yet annotated |
Frequently Asked Questions
What is the expected molecular weight of recombinant PKARIIA on SDS-PAGE and Western blot?
Recombinant human PKARIIA (REC-PKARIIA) runs at approximately 45–47 kDa on reducing SDS-PAGE, consistent with the 401-amino-acid PRKAR2A sequence (UniProt P12367, calculated MW ~46.2 kDa). HEK293-derived protein may migrate slightly above the calculated mass due to mammalian post-translational modifications, including phosphorylation. Purity is >95% by SDS-PAGE; a single predominant band at this position is expected. If you are running a Western blot positive control alongside RP-PKARIIA antibody, load 20–50 ng per lane for a clean, non-saturating signal.
Which isoform or processing form of PKARIIA does REC-PKARIIA correspond to — is it full-length?
REC-PKARIIA corresponds to the full-length human RIIα regulatory subunit, residues 1–401 (PRKAR2A, UniProt P12367), with no truncation or propeptide removal. It is expressed in HEK293 cells, so it retains mammalian phosphorylation relevant to the native dimerization domain (DD) and both tandem cyclic-nucleotide-binding (CNB-A and CNB-B) domains. No signal peptide or cleavage event is expected for this cytosolic/membrane-associated regulatory subunit; the recombinant protein represents the mature, full-length form.
Does recombinant PKARIIA bind cAMP and can I use it in a cAMP-binding or competition assay?
Yes. REC-PKARIIA retains functional CNB-A and CNB-B domains and is suitable for cAMP-binding assays, including fluorescence polarization competition formats and radioligand ([³H]cAMP) displacement assays. The Kd for cAMP at the high-affinity CNB-B domain is in the low-nanomolar range (~1–10 nM); CNB-A has moderately lower affinity. For competition assays, a working concentration of 50–200 nM REC-PKARIIA is a practical starting point, titrated against cAMP analogs or test compounds. Assay in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, supplemented with 1 mM DTT to maintain cysteine integrity.
What buffer and conditions should I use for PKARIIA activity or interaction assays?
REC-PKARIIA is supplied in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol and is compatible with standard PKA assay buffers at pH 7.2–7.5. For holoenzyme reconstitution or AKAP pull-down assays, dilute into binding buffer containing 20 mM HEPES pH 7.4, 150 mM NaCl, 2 mM MgCl₂, and 0.1% Tween-20. Avoid chelating agents (EDTA >1 mM) that may interfere with divalent-ion-dependent interactions. For cAMP-release kinase activation assays, supplement with 1–10 µM cAMP and 1 mM DTT. Equilibrate protein on ice for 5 minutes before use after thaw.
What starting concentration of recombinant PKARIIA should I use to reconstitute a PKA holoenzyme in vitro?
For in vitro holoenzyme reconstitution with catalytic subunit (PKA-Cα), use a 1:1 to 2:1 molar ratio of REC-PKARIIA to catalytic subunit, starting at 100–500 nM each. RIIα forms a (RIIα)₂(Cα)₂ tetramer; confirm assembly by native PAGE or size-exclusion chromatography. Working concentrations of 200 nM REC-PKARIIA are sufficient for most SPR or BLI-based AKAP interaction studies. For substrate phosphorylation inhibition assays using a kemptide-type substrate (Kemptide: LRRASLG), titrate REC-PKARIIA from 50 nM to 2 µM to establish full inhibition curves and determine apparent IC₅₀ relative to your catalytic subunit concentration.
Can I use REC-PKARIIA as a positive control for Western blot with the RP-PKARIIA antibody?
Yes — REC-PKARIIA is the validated positive control antigen for the matched rabbit polyclonal antibody RP-PKARIIA (/anti-pka-riia-rabbit-polyclonal-antibody). Load 20–50 ng of recombinant protein per lane on a standard 10–12% SDS-PAGE gel; expect a band at ~45–47 kDa. RP-PKARIIA is validated for Western blot detection of this band. Running REC-PKARIIA alongside cell lysates (e.g., HEK293, HeLa) provides a clean size reference and confirms antibody functionality, which is particularly useful when validating new lot numbers or troubleshooting background issues in complex tissue lysates.
How much recombinant PKARIIA should I load for Western blot positive control alongside RP-PKARIIA antibody?
20–50 ng per lane gives a strong, non-saturating band at ~45–47 kDa with RP-PKARIIA at standard working dilutions (typically 1:500–1:2,000 for Western blot, per the RP-PKARIIA datasheet). If your cell lysate lane is loaded at 20–30 µg total protein, 20 ng recombinant is sufficient to produce a clearly visible positive control band without overwhelming adjacent lanes. Dilute REC-PKARIIA into Laemmli sample buffer immediately before boiling; the 10% glycerol in the storage buffer is compatible with standard sample preparation and does not require buffer exchange.
What is the shelf life of REC-PKARIIA and how should I handle it after thawing to preserve activity?
REC-PKARIIA is stable for at least 12 months from date of receipt when stored at -20°C in single-use aliquots. After thawing, keep on ice and use within 4–6 hours; repeated freeze-thaw cycles progressively reduce binding activity and should be strictly avoided. Do not dilute into water — maintain protein in the supplied 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol buffer or an equivalent formulation. If working stocks are needed, add BSA (0.1 mg/mL) as a carrier stabilizer and store at 4°C for up to 72 hours. Endotoxin is <0.1 EU/µg by LAL assay.
Validation imagery coming soon
Western blot validation figures for REC-PKARIIA will be published here as they are produced in-house.
If you would like to see existing validation data for this antibody before publication, request a sample copy.