Anti-von Willebrand Factor Rabbit Polyclonal Antibody

Frequently Asked Questions

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

PKA-RIIB (also known as PRKAR2B) typically migrates at approximately 46 kDa on reducing SDS-PAGE, corresponding to the full-length regulatory subunit. Some labs report doublets or minor bands near 50 kDa depending on post-translational modifications or splice variants. Human tissue lysates from brain, adipose, or adrenal gland usually show the cleanest signal, as RIIB is highly expressed in these tissues. If you see additional bands, verify lysate quality and consider phosphatase treatment to collapse phosphorylated species. Starting dilution of 1:1000 works for most enriched samples.

How does PKA-RIIB differ from PKA-RIα and other regulatory subunit isoforms?

PKA-RIIB is one of four mammalian regulatory subunits (RIα, RIβ, RIIα, RIIB) that determine cAMP sensitivity and subcellular localization of protein kinase A holoenzymes. RIIB exhibits higher cAMP affinity than type I subunits and contains an N-terminal domain that mediates association with A-kinase anchoring proteins, directing compartmentalized signaling. Tissue distribution also differs: RIIB is abundant in brain, adipose, and endocrine tissues, whereas RIα is more ubiquitous. Antibody cross-reactivity between isoforms is common, so include isoform-specific controls or knockdown lysates if selectivity is critical to your experiment.

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

This antibody is validated for human PKA-RIIB. Cross-reactivity with mouse and rat is predicted based on high sequence homology in the immunogen region, but we have not formally validated it in rodent lysates. If you plan to use mouse or rat samples, run a pilot blot with brain or adipose lysate alongside a human positive control. Many researchers report successful detection in rodent tissues at the recommended 1:1000 dilution, but band intensity and specificity should be confirmed in your hands before committing to a full experiment.

What sample preparation is recommended for detecting PKA-RIIB by Western blot?

Use RIPA or a Triton X-100-based lysis buffer supplemented with protease and phosphatase inhibitors, as PKA-RIIB is a cytosolic and membrane-associated protein. Load 20–40 µg total protein per lane for whole-cell lysates; enriched fractions or tissues with high RIIB expression may require less. Avoid boiling samples excessively, as prolonged heating can cause aggregation of some regulatory subunits. Include a human brain or adipose tissue lysate as a positive control. If signal is weak, verify antibody dilution, increase exposure time, or consider enriching the cytosolic fraction by differential centrifugation.

What controls should I include when using this PKA-RIIB antibody?

Include a known RIIB-expressing tissue or cell line as a positive control—human brain lysate, differentiated adipocytes, or adrenal gland work well. A negative control could be a tissue with low or absent RIIB expression, though complete absence is rare. For specificity, consider siRNA or shRNA knockdown of PRKAR2B in your cell model; loss of the 46 kDa band confirms on-target binding. A no-primary-antibody control helps rule out non-specific secondary binding. If you suspect isoform cross-reactivity, run lysates from cells overexpressing individual PKA-R isoforms in parallel.

Can I use this antibody for immunofluorescence or immunohistochemistry on PKA-RIIB?

Triple Point Biologics antibodies are validated for Western blot and immunohistochemistry applications. This rabbit polyclonal should be compatible with IHC on formalin-fixed paraffin-embedded sections, though you will need to optimize antigen retrieval and blocking conditions for your tissue type. For immunofluorescence, start with a 1:100 to 1:200 dilution and include a peptide-competition control or RIIB-knockdown cells to confirm specificity. Cytosolic and perinuclear staining patterns are typical for RIIB. We recommend piloting a small batch before scaling up, as fixation and permeabilization can influence epitope accessibility.

How should I store the PKA-RIIB antibody and how long does it remain stable?

Store the antibody at –20°C in small aliquots to avoid repeated freeze-thaw cycles, which can reduce titer and increase aggregation. Once thawed, an aliquot can be kept at 4°C for up to one month if supplemented with 0.02–0.05% sodium azide or a commercial stabilizer. Do not store diluted antibody for extended periods; prepare working dilutions fresh in blocking buffer for each experiment. Under proper storage, the antibody typically retains activity for at least two years from the date of manufacture. If you notice declining signal or increased background over time, prepare a fresh aliquot.