Anti-PKA-RIA Rabbit Polyclonal Antibody

Frequently Asked Questions

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

The type I-alpha regulatory subunit of PKA typically migrates at approximately 43 kDa on SDS-PAGE, corresponding to the full-length PRKAR1A protein. You may observe doublets or slight shifts due to phosphorylation at multiple regulatory sites or N-terminal processing events. In some cell types with high PKA activity, you might detect minor proteolytic fragments below 43 kDa. Always run a positive control lysate from tissues or cells with high cAMP signaling activity, such as brain, heart, or endocrine tissues, to confirm the expected band pattern before analyzing experimental samples.

How do I distinguish PKA-RIA from PKA-RIB on a Western blot?

PKA-RIA (PRKAR1A gene product, ~43 kDa) and PKA-RIB (PRKAR1B gene product, ~49 kDa) migrate at different molecular weights, which aids identification. This antibody was raised against human PKA-RIA and is validated specifically for that isoform. If you need to assess both type I regulatory subunits simultaneously, run separate blots with isoform-specific antibodies rather than relying on band size alone, since post-translational modifications can alter migration. RIA predominates in most somatic tissues, whereas RIB shows enrichment in brain, adipose, and certain cancer cell lines.

Does this PKA-RIA antibody cross-react with mouse or rat samples?

The antibody is validated in human samples and shows predicted cross-reactivity with mouse and rat based on sequence homology between human PRKAR1A and rodent Prkar1a orthologs, which share greater than 95 percent amino acid identity in the immunogen region. However, you should confirm reactivity and optimal dilution empirically with your specific rodent tissue or cell lysate. Start at the recommended 1:1000 dilution for Western blot and adjust based on signal strength. Brain, heart, and testis lysates serve as useful positive controls for rodent samples due to high endogenous PKA-RIA expression.

What dilution should I start with for immunofluorescence with this PKA-RIA antibody?

Although this antibody is validated for Western blot at 1:1000, immunofluorescence typically requires higher antibody concentrations. A reasonable starting dilution for IF is between 1:100 and 1:200, then titrate based on signal-to-noise ratio in your specific cell type. PKA-RIA localizes primarily to the cytoplasm and cell membrane under basal conditions, so expect diffuse cytoplasmic staining with possible membrane enrichment. Include a secondary-antibody-only control to assess background, and consider treating cells with forskolin or IBMX to elevate cAMP and potentially redistribute PKA subunits if studying dynamic localization.

What are good positive and negative control tissues for PKA-RIA Western blots?

PKA-RIA is widely expressed, making true negative tissues rare. Brain, heart, skeletal muscle, and endocrine tissues such as adrenal or pancreas show robust expression and serve as excellent positive controls. For a relative negative control, consider terminally differentiated cells with lower proliferative signaling, though complete absence is uncommon. Alternatively, use siRNA or CRISPR knockdown cell lines targeting PRKAR1A as a functional negative control to confirm antibody specificity. Including both a high-expression tissue and a knockdown sample on the same blot provides the most convincing validation of signal specificity.

Can I use this antibody to detect PKA-RIA in immunohistochemistry on FFPE sections?

this antibody is validated for Western blot; IHC validation is in progress applications, making it suitable for formalin-fixed paraffin-embedded tissue sections. Antigen retrieval is typically required; citrate buffer (pH 6.0) heat-mediated retrieval is a standard starting method for PKA regulatory subunits. Expect cytoplasmic and membranous staining patterns in epithelial and stromal cells. Optimal antibody dilution for IHC often differs from Western blot and may range from 1:50 to 1:200. Titrate on your specific tissue type and include appropriate positive control sections such as normal brain or cardiac tissue to establish staining conditions before scoring experimental samples.

How should I store this PKA-RIA antibody and what is the typical shelf life?

Store the antibody at -20°C in single-use aliquots to avoid repeated freeze-thaw cycles, which can reduce titer and increase aggregation. The product ships as 100 µg and typically remains stable for at least 12 months from receipt when stored correctly. If you use the antibody frequently, a working aliquot can be kept at 4°C for up to one month; add sodium azide (0.02 to 0.05 percent final) as a preservative if storing diluted stocks. Avoid prolonged exposure to room temperature. Centrifuge briefly before use if you observe particulates, which can cause high background in immunofluorescence.

What sample preparation considerations are important for detecting PKA-RIA by Western blot?

Use fresh or snap-frozen lysates when possible, as PKA regulatory subunits can undergo proteolysis during prolonged sample handling. Include protease inhibitors (AEBSF, aprotinin, leupeptin) and phosphatase inhibitors (sodium fluoride, sodium orthovanadate) in lysis buffer, since PKA-RIA is phosphorylated at multiple sites that affect its electrophoretic mobility. RIPA or NP-40 lysis buffers work well for extracting cytoplasmic and membrane-associated pools. Load 20 to 40 µg total protein per lane as a starting point. Avoid boiling samples excessively; five minutes at 95°C is sufficient and reduces aggregation that can smear the 43 kDa band.