Anti-PKA-RIB Rabbit Polyclonal Antibody

Frequently Asked Questions

What is the expected molecular weight of PKA-RIB on Western blot?

PKA-RIB (PRKAR1B) runs at approximately 46 kDa on Western blot under reducing conditions, corresponding to the full-length regulatory subunit I-beta. You may occasionally observe a doublet or minor higher molecular weight bands around 50-52 kDa, which can represent post-translational modifications such as phosphorylation. The protein is encoded by PRKAR1B and consists of 381 amino acids in humans. When probing brain or adipose tissue lysates, where PRKAR1B is highly expressed, you should see a clean band at 46 kDa. Always include a positive control lysate from these tissues to confirm antibody performance.

What dilution should I start with for PKA-RIB Western blot?

Start with a 1:1000 dilution in blocking buffer (5% non-fat milk or BSA in TBST) for Western blot applications. This is our validated dilution for this rabbit polyclonal antibody. Incubate overnight at 4°C for optimal signal. Depending on your tissue lysate and expression level, you may need to titrate between 1:500 and 1:2000. Brain and adipose lysates, which express high endogenous PRKAR1B levels, typically work well at 1:1000. For tissues with lower expression, start at 1:500 and optimize from there. Use 20-30 µg total protein per lane as a starting point.

Does this PKA-RIB antibody cross-react with other PKA regulatory subunits?

This antibody is raised against PKA-RIB (PRKAR1B) and is designed to specifically recognize the type I-beta regulatory subunit. There are four PKA regulatory subunits (RIα, RIβ, RIIα, RIIβ) with partial sequence homology, particularly within conserved cAMP-binding domains. Cross-reactivity with RIα (PRKAR1A) is possible given both are type I subunits, though they differ in molecular weight (RIα is approximately 43 kDa). We have validated specificity for human PRKAR1B. If you are working in a system expressing multiple R subunits, run parallel blots with isoform-specific controls or use siRNA knockdown to confirm band identity.

Will this antibody work in mouse or rat samples?

This antibody is validated for human PKA-RIB. Cross-reactivity with mouse and rat is predicted based on sequence homology between human and rodent PRKAR1B orthologs, which share greater than 90% identity at the protein level. Mouse PRKAR1B (UniProt Q921L9) is highly conserved in the immunogenic regions. However, cross-reactivity has not been formally validated by us. If working with mouse or rat tissue, we recommend testing the antibody at 1:500 to 1:1000 on brain or adipose lysates first, as these tissues show the highest PRKAR1B expression. Include a human lysate positive control on the same blot for comparison.

What are good positive and negative control tissues for PKA-RIB?

PKA-RIB is most abundant in brain tissue and adipose tissue, making these ideal positive controls. Neuronal cells and adipocytes rely heavily on compartmentalized cAMP signaling where PRKAR1B plays a key role in membrane-proximal PKA anchoring. For cell lines, SH-SY5Y (neuroblastoma) or primary neurons are good positive controls. As a negative control, consider tissues or cell lines with very low PRKAR1B expression, though true nulls are rare since PKA regulatory subunits are broadly expressed. A more practical negative control is lysate pretreated with siRNA against PRKAR1B or a PRKAR1B knockout cell line if available.

Can I use this PKA-RIB antibody for immunofluorescence or IHC?

Yes, Triple Point Biologics antibodies are validated for immunofluorescence and immunohistochemistry in addition to Western blot. For IF, start with a 1:100 to 1:200 dilution and optimize based on signal intensity and background. PKA-RIB localizes primarily to the plasma membrane, so expect peripheral or cortical staining patterns in cells with high expression. For IHC on paraffin-embedded tissue, antigen retrieval is typically required; citrate buffer (pH 6.0) heat-mediated retrieval is a standard starting point. Brain and adipose tissue sections serve as excellent positive controls. Titrate the antibody concentration to balance specific signal against background for your fixation and processing conditions.

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

Store the antibody at -20°C in its original packaging. Avoid repeated freeze-thaw cycles, which can reduce antibody activity over time. For frequent use, consider making single-use aliquots (10-20 µL) upon receipt and storing them at -20°C or -80°C. Once thawed, an aliquot can be kept at 4°C for up to one month if supplemented with 0.02% sodium azide as a preservative. Do not add azide if you plan to use the antibody for applications involving HRP-conjugated detection, as azide inhibits peroxidase. Under proper storage at -20°C, the antibody remains stable for at least two years from the date of receipt.

Why am I seeing multiple bands with PKA-RIB antibody on Western blot?

Multiple bands can arise from several sources. PKA-RIB undergoes phosphorylation and other post-translational modifications that shift its mobility slightly, producing doublets or bands in the 46-52 kDa range. Proteolytic degradation during sample preparation can generate lower molecular weight fragments; ensure you use fresh protease inhibitors and keep samples on ice. Cross-reactivity with PKA-RIα (43 kDa) is also possible given sequence similarity between type I regulatory subunits. To troubleshoot, run a positive control (brain lysate) side-by-side, confirm your sample prep includes protease and phosphatase inhibitors, and consider treating lysate with lambda phosphatase to collapse phosphorylation-dependent bands into a single species.