Anti-PRKAR2A (PKA-RIIA) Rabbit Polyclonal Antibody

Frequently Asked Questions

What is the expected molecular weight for PKA-RIIA on Western blot?

PKA-RIIA (PRKAR2A) runs at approximately 46 kDa on SDS-PAGE, corresponding to the 401-amino acid regulatory subunit. You may observe a tight doublet or minor shifts depending on post-translational modifications, particularly phosphorylation states that can vary with cAMP signaling status. If you see bands significantly higher than 46 kDa, consider that PKA-RIIA can exist in dimeric complexes under non-reducing conditions or incomplete denaturation. Always run samples with fresh reducing agent and adequate boiling time. A positive control lysate from cAMP-stimulated cells will help confirm the expected migration pattern.

How does PKA-RIIA differ from other PKA regulatory subunits on Western blot?

PKA-RIIA (type II-alpha, 46 kDa) is the most abundant type II regulatory subunit in many cell types and differs from RIα (43 kDa) and RIβ (45 kDa) by size and subcellular distribution. Type II subunits like RIIA bind A-kinase anchoring proteins (AKAPs) and localize to membranes and cytoskeleton, whereas type I subunits are more diffusely cytoplasmic. On a blot, if you see multiple bands between 43-52 kDa, you may be detecting cross-reactivity with RI isoforms or the closely related RIIβ (54 kDa). Isoform-specific antibodies and careful molecular-weight assessment are essential for disambiguation.

What dilution should I start with for PKA-RIIA Western blots?

We recommend starting at 1:1000 dilution for Western blot with this rabbit polyclonal PKA-RIIA antibody, based on validation in human lysates. Expression levels vary widely by tissue; heart, brain, and adipose typically show robust RIIA signal, while some transformed cell lines express lower levels. If your signal is weak, try 1:500 or increase protein load to 30-50 micrograms per lane before increasing antibody concentration. Conversely, if you see high background, dilute to 1:2000. Titration against your specific lysate is the most reliable way to balance signal and specificity.

Which sample preparation steps are critical for detecting PKA-RIIA?

PKA-RIIA is relatively stable, but proper lysis and protease inhibition are critical because the holoenzyme complex is sensitive to endogenous proteases and phosphatases. Use a lysis buffer containing phosphatase inhibitors if you are studying cAMP-dependent regulation or phosphorylation-dependent band shifts. Avoid repeated freeze-thaw cycles, which can promote regulatory subunit degradation. Because RIIA associates with AKAPs and cytoskeletal structures, mechanical lysis or brief sonication can improve extraction from particulate fractions. For cytoplasmic enrichment, differential centrifugation will concentrate RIIA in the soluble and light-membrane fractions rather than nuclear pellets.

Is this PKA-RIIA antibody validated in species other than human?

This antibody is validated for human PKA-RIIA. Cross-reactivity with mouse and rat is predicted based on high sequence homology in the immunogen region (typically greater than 90 percent identity across mammalian PRKAR2A orthologs), but we have not independently validated reactivity in rodent lysates. If you are working with mouse or rat samples, plan to run a positive control such as heart or brain lysate alongside your experimental samples and be prepared to optimize dilution. Reactivity in more distant species such as zebrafish or Xenopus is unlikely without additional validation.

What are good positive and negative controls for PKA-RIIA experiments?

Human heart, skeletal muscle, and brain lysates are excellent positive controls due to high endogenous RIIA expression. HEK293 and HeLa cells express moderate levels and are convenient for cultured-cell controls. For a functional control, treat cells with forskolin or IBMX to elevate cAMP; this will not change RIIA protein levels acutely but confirms your signaling pathway is intact. Negative controls include PRKAR2A knockout cell lines if available, or siRNA knockdown with at least 72-hour depletion. Pre-incubation of the antibody with the immunizing peptide, if provided, can also serve as a blocking control to verify specificity.

How should I store this PKA-RIIA antibody to maintain activity?

Store the antibody at -20°C in single-use aliquots to avoid repeated freeze-thaw cycles, which degrade antibody binding over time. If you use the antibody frequently, a working aliquot can be kept at 4°C for up to one month with 0.02 percent sodium azide as preservative; check the product datasheet for formulation details. Do not store diluted antibody in blocking buffer for more than a few days, as carrier proteins can promote bacterial growth and reduce titer. Polyclonal antisera are generally robust, but long-term storage beyond one year at -20°C may benefit from transfer to -80°C for maximum stability.

Can this antibody detect PKA-RIIA in immunofluorescence or immunohistochemistry?

Triple Point Biologics antibodies are validated for Western blot; this PKA-RIIA antibody is suitable for both applications. For immunofluorescence, start with a 1:100 to 1:200 dilution and expect cytoplasmic and perinuclear staining that reflects RIIA association with AKAPs and cytoskeletal anchoring sites. Antigen retrieval with citrate buffer (pH 6) is typically required for paraffin-embedded sections. Because RIIA is broadly expressed, include a no-primary-antibody control and compare staining intensity across tissues known to have high versus low expression. Frozen sections often yield cleaner signal than FFPE for this target.