PKARIIB (Recombinant)

Frequently Asked Questions

What is the expected molecular weight of recombinant PKARIIB on SDS-PAGE?

Recombinant PKARIIB (REC-PKARIIB) runs at approximately 52 kDa under reducing SDS-PAGE conditions, consistent with the full-length regulatory subunit IIβ of PKA. Produced in HEK293 cells, this preparation may appear as a slightly diffuse band compared to bacterially expressed protein due to mammalian post-translational modifications. Purity is >90% by SDS-PAGE. When loading a positive control lane alongside RP-PKARIIB Western blot detection, 50–100 ng is sufficient to resolve a clean band at the expected molecular weight.

Which isoform or processing form of PKARIIB does this recombinant represent?

REC-PKARIIB corresponds to the full-length human PKARIIB (UniProt P13861), the type IIβ regulatory subunit of cAMP-dependent protein kinase A. This isoform is predominantly expressed in brain, adipose tissue, and adrenal gland and is distinguished from RIIα by its unique N-terminal dimerization/docking (D/D) domain sequence. The protein is expressed without truncation in HEK293 cells, preserving the intact D/D domain, the autophosphorylation site at Ser96, and both cAMP-binding domains (CNB-A and CNB-B), which are required for authentic holoenzyme regulation.

Does PKARIIB get cleaved by caspases or other proteases, and does this recombinant reflect that?

PKARIIB is a known caspase-3 substrate; cleavage at Asp299 generates an ~33 kDa C-terminal fragment during apoptosis, releasing constitutive kinase activity from regulatory control. REC-PKARIIB is produced as the intact, uncleaved full-length form and is not pre-processed. If your experiment models apoptotic PKA dysregulation, you would need to cleave this preparation in vitro with recombinant caspase-3. The intact full-length form is the appropriate antigen for benchmarking against RP-PKARIIB (anti-PKARIIB rabbit polyclonal antibody), which is raised against residues spanning both CNB domains.

What activity assay is recommended for recombinant PKARIIB, and what substrate should I use?

PKARIIB is a regulatory, not catalytic, subunit — it does not phosphorylate substrates directly. Functional assays therefore measure its inhibitory activity toward the PKA catalytic subunit (PKAc). A standard approach uses a Kemptide (LRRASLG) phosphorylation assay: pre-assemble PKA holoenzyme by incubating REC-PKARIIB with recombinant PKAcα at a 2:1 molar ratio (RII:C), then titrate cAMP (0.01–10 µM) to dissociate the complex and de-repress catalytic activity. Phosphotransfer to Kemptide is then quantified by luminescent or radioactive ATP consumption assay, yielding EC50 values for cAMP in the 100–300 nM range.

What buffer conditions work best for PKARIIB holoenzyme assembly and cAMP-binding assays?

REC-PKARIIB is supplied in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol. For holoenzyme assembly and cAMP-binding assays, dilute into assay buffer containing 20 mM MOPS pH 7.0, 100 mM NaCl, 1 mM EDTA, 0.1 mg/mL BSA, and 1 mM DTT. The EDTA chelates adventitious divalent cations that can interfere with cAMP binding affinities. Glycerol from the storage buffer (carried over at <1% final) is generally well tolerated. Avoid prolonged incubation above 30°C; keep assembled holoenzyme on ice until assay initiation to minimize spontaneous dissociation.

What starting concentration of recombinant PKARIIB should I use in a PKA holoenzyme reconstitution experiment?

A practical starting point is 200–500 nM REC-PKARIIB combined with equimolar to 2× molar excess over recombinant PKAcα in your reconstitution buffer. At these concentrations, holoenzyme formation is robust within 30 minutes on ice. For cAMP-competition binding assays (e.g., using [³H]-cAMP or fluorescent cAMP analogs), 50–100 nM REC-PKARIIB per well in a 96-well format is typical. Protein concentration of the supplied stock is stated on the Certificate of Analysis; verify by A280 or BCA before use, as glycerol in the buffer can interfere with direct absorbance readings.

Can I use REC-PKARIIB as a positive control for Western blot with the RP-PKARIIB antibody?

Yes — REC-PKARIIB is the intended positive control antigen for RP-PKARIIB (/anti-pka-riib-rabbit-polyclonal-antibody). Load 50–100 ng per lane under reducing conditions; this produces a strong, clean band at ~52 kDa with RP-PKARIIB at 1:1,000–1:2,000 dilution in 5% non-fat milk/TBST. Because both products originate from the same TPB lab, immunoreactivity is validated rather than predicted. This pairing is particularly useful when establishing PKARIIB detection in a new tissue lysate or cell line where endogenous expression levels are uncertain.

How much recombinant PKARIIB should I load for a Western blot positive control lane alongside cell lysates?

50 ng of REC-PKARIIB per lane is a reliable starting point alongside cell lysates when probing with RP-PKARIIB. This amount sits well within the linear dynamic range of standard chemiluminescence detection and prevents the recombinant band from overwhelming endogenous signals in adjacent lanes. If your lysate background is high or you are titrating antibody conditions, run a dilution series of 25, 50, and 100 ng in parallel. Under reducing SDS-PAGE, expect a single band at ~52 kDa; absence of higher-MW smearing confirms the preparation has not aggregated.

How should I handle, dilute, and store recombinant PKARIIB to maintain activity long-term?

REC-PKARIIB is supplied as single-use aliquots in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol and should be stored at -20°C. Avoid repeated freeze-thaw cycles, which accelerate aggregation and reduce cAMP-binding competence. On the day of use, thaw on ice and dilute into pre-chilled assay buffer; do not vortex — mix gently by pipetting. Working dilutions should be prepared fresh and kept on ice for no longer than 4–6 hours. Under recommended storage, activity is stable for at least 12 months from the manufacturing date listed on the Certificate of Analysis.