PKARIA (Recombinant)

Frequently Asked Questions

What molecular weight band should I expect for PKARIA (Recombinant) on SDS-PAGE or Western blot?

The full-length PKARIA protein (381 amino acids, UniProt Q9DBC7) has a calculated molecular weight of approximately 43 kDa. On reducing SDS-PAGE, this HEK293-expressed recombinant typically migrates between 43–47 kDa; slight upward shift from post-translational modifications introduced in the mammalian expression system is normal. Purity is >95% by SDS-PAGE, so a clean, predominant band in that range is expected. If you observe a doublet, this likely reflects phosphorylation heterogeneity rather than a contaminating species.

Is PKARIA (Recombinant) the full-length protein or a truncated regulatory subunit fragment?

This recombinant represents the full-length human PRKAR1A-encoded protein spanning all 381 amino acids, including both tandem cyclic-nucleotide-binding domains (CNB-A and CNB-B) and the dimerization/docking (D/D) domain at the N-terminus. No truncations were introduced. Because it is expressed in HEK293 cells, it carries native-relevant post-translational modifications — important for studies reconstituting intact PKA holoenzyme or characterizing regulatory subunit–catalytic subunit interaction surfaces at physiological fidelity.

What is PKARIA's function in the PKA holoenzyme and how does cAMP trigger its release?

PKARIA is the type I-alpha regulatory subunit that holds the PKA catalytic subunit (Cα or Cβ) in an inactive R2C2 holoenzyme complex under basal cAMP conditions. When intracellular cAMP rises, two cAMP molecules bind cooperatively to the CNB-A and CNB-B domains of each PKARIA subunit, inducing a conformational change that releases active catalytic subunits. This recombinant retains both functional CNB domains, making it directly suitable for fluorescence polarization or ITC-based cAMP-binding affinity measurements and holoenzyme reconstitution assays.

What assay format and starting concentration should I use to measure cAMP binding to recombinant PKARIA?

For fluorescence polarization cAMP-binding assays using a fluorescent cAMP analog (e.g., 8-[Fluo]-cAMP), a starting PKARIA concentration of 50–200 nM works well for Kd determination, given the reported Kd values of ~50–300 nM depending on CNB domain and analog used. Run the assay in 50 mM Tris-HCl pH 7.5, 150 mM NaCl — matching the storage buffer — to avoid rebuffering artifacts. For ITC, 5–10 µM protein in the cell with 100–200 µM cAMP in the syringe is a practical starting point. Titrate protein concentration empirically if signal is low.

What buffer conditions are recommended for PKA holoenzyme reconstitution using this recombinant PKARIA?

The storage buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol) is compatible with most in vitro reconstitution protocols. For holoenzyme assembly, mix PKARIA with the catalytic subunit (Cα) at an equimolar R:C ratio (typically 0.5–2 µM each) in the same buffer supplemented with 1 mM DTT and 1 mM MgCl₂; incubate 30 min on ice before use. Avoid stripping cAMP from the preparation with high-salt washes before confirming holoenzyme formation by native PAGE or SEC, as the recombinant may carry residual nucleotide from expression.

Can I use PKARIA (Recombinant) as a positive control for Western blot with the matched RP-PKARIA antibody?

Yes — this is one of the primary intended uses. The recombinant PKARIA (REC-PKARIA) and the matched rabbit polyclonal antibody (RP-PKARIA) are produced and validated in the same lab, with guaranteed Western blot compatibility. Load 10–50 ng of recombinant per lane alongside your cell lysate samples; the antibody should detect a clean band at 43–47 kDa. This loading range keeps the recombinant lane within the linear detection window without overwhelming the signal from endogenous PKARIA in your lysate lanes. See the RP-PKARIA product page for recommended antibody dilutions.

How much PKARIA (Recombinant) should I load for a Western blot positive control lane?

10–50 ng per lane is the recommended range when using the matched RP-PKARIA rabbit polyclonal antibody (SKU: RP-PKARIA). Start at 25 ng if you are unsure of your detection system's sensitivity. At >90% purity, virtually all loaded protein contributes to the target band at 43–47 kDa, so this amount produces a robust, interpretable signal without requiring large volumes of your aliquot. If co-running with cell lysates, loading 25 ng recombinant alongside 20–30 µg total lysate gives a comparable signal intensity for most anti-PKARIA antibody dilutions.

How should I store and handle PKARIA (Recombinant) to preserve activity across multiple experiments?

Store at -20°C in the supplied single-use aliquots. The 10% glycerol in the storage buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl) provides cryoprotection and supports stability through at least 12 months when stored correctly. Avoid repeated freeze-thaw cycles — each cycle risks partial unfolding of the CNB domains, which can reduce cAMP-binding capacity and holoenzyme reconstitution efficiency. On the day of use, thaw on ice, briefly centrifuge at 10,000 × g to pellet any aggregates, and keep on ice during the experiment. Do not dilute the working stock further than necessary until immediately before use.