PRKAR2B Antibody

What are the primary applications for PRKAR2B antibodies?

PRKAR2B antibodies are validated for multiple experimental techniques including:

The optimal dilution should be determined experimentally for each specific application and sample type .

What tissues and cell lines show positive PRKAR2B expression?

PRKAR2B antibodies have demonstrated positive reactivity in:

• Tissues: Human testis, human stomach, human ovary, mouse brain, rat brain, mouse testis, mouse eye

• Cell lines: Y79, Neuro-2a, SH-SY5Y

• Sample types: Brain lysates, testis lysates, various neuronal cell lines

For optimal results when working with new tissue or cell types, preliminary experiments to confirm expression are recommended.

What is the expected molecular weight for PRKAR2B detection?

PRKAR2B has a calculated molecular weight of 46 kDa (418 amino acids), though the observed molecular weight in Western blot applications typically ranges from 46-50 kDa depending on post-translational modifications and the specific tissue or cell type being examined .

How should I optimize antigen retrieval for PRKAR2B IHC?

For optimal PRKAR2B detection in formalin-fixed, paraffin-embedded tissues:

• Primary recommendation: Use TE buffer at pH 9.0 for heat-induced epitope retrieval

• Alternative method: Citrate buffer at pH 6.0 may be effective for certain tissue types

• Incubation time: 15-20 minutes at 95-100°C followed by cooling to room temperature

• Controls: Include positive control tissues (human stomach or testis) and negative controls

Signal intensity and specificity should be evaluated against appropriate controls to determine the optimal retrieval conditions for your specific tissue samples.

What blocking and incubation conditions are recommended for PRKAR2B antibody applications?

For all applications, optimizing antibody concentration through titration experiments is strongly recommended to achieve the best signal-to-noise ratio .

How do I troubleshoot non-specific binding or background issues with PRKAR2B antibodies?

When encountering high background or non-specific binding:

• Increase blocking time/concentration: Use 5-10% BSA or normal serum from the same species as the secondary antibody

• Optimize antibody dilution: Further dilute the primary antibody beyond recommended ranges

• Add detergents: Include 0.1-0.3% Triton X-100 or 0.05-0.1% Tween-20 in washing buffers

• Implement additional washing steps: Increase number and duration of washes

• Use absorption controls: Pre-incubate antibody with recombinant PRKAR2B protein

• Evaluate cross-reactivity: Test the antibody with known negative controls

If background persists, consider using monoclonal antibodies like EP2649Y (ab75993) or EP2648 (ab75996) which may offer improved specificity compared to polyclonal alternatives .

How can I validate PRKAR2B antibody specificity?

Multiple validation approaches should be employed:

• Positive/negative controls: Test tissues with known expression patterns (brain, testis as positive; depleted samples as negative)

• Knockdown/knockout verification: Use siRNA or CRISPR to generate PRKAR2B-deficient cells

• Peptide competition: Pre-incubate with immunizing peptide to confirm specific binding

• Multiple antibody comparison: Use antibodies targeting different PRKAR2B epitopes

• Molecular weight confirmation: Verify observed band matches predicted 46-50 kDa

• Multiple techniques: Confirm results across WB, IHC, and IF applications

Research has shown that knockdown of PRKAR2B in specific contexts leads to observable phenotypes, which can serve as functional validation of antibody specificity .

What is the role of PRKAR2B in reproductive biology and development?

Studies using validated PRKAR2B antibodies have revealed:

• PRKAR2B is highly expressed in ovarian tissues, particularly in growing follicles

• During oocyte maturation, PRKAR2B expression peaks during metaphase I (MI) and decreases at metaphase II (MII)

• RNAi-mediated PRKAR2B suppression results in MI-stage arrest during oocyte development

• PRKAR2B-deficient oocytes exhibit abnormal spindle formation and chromosome aggregation

• PRKAR2B appears to regulate the pentose phosphate pathway (PPP) in oocytes

These findings suggest PRKAR2B is critical for proper oocyte maturation by controlling spindle formation and PPP-mediated metabolism .

How is PRKAR2B implicated in cancer research?

Recent research has identified PRKAR2B as a potential oncogenic factor:

• PRKAR2B is overexpressed in castration-resistant prostate cancer (CRPC)

• Functional validation experiments show PRKAR2B promotes CRPC cell proliferation and invasion

• PRKAR2B inhibits CRPC cell apoptosis

• Transcriptome and GO enrichment analyses indicate PRKAR2B primarily accelerates cell cycle processes

• PRKAR2B modulates multiple cell cycle genes including CCNB1, MCM2, PLK1, and AURKB

These findings suggest PRKAR2B may be a promising therapeutic target for CRPC treatment and further investigation .

What storage and handling protocols optimize PRKAR2B antibody performance?

To maintain antibody stability and performance:

Most PRKAR2B antibodies are supplied in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3. Some formulations may contain 0.1% BSA for additional stability. For optimal results, avoid repeated freeze-thaw cycles .

How should I select between monoclonal and polyclonal PRKAR2B antibodies?

The choice depends on your experimental objectives:

Monoclonal Antibodies (e.g., EP2649Y, EP2648)

• Advantages: Higher specificity, reduced batch-to-batch variation, consistent results

• Best for: Quantitative analysis, detecting specific epitopes, long-term studies

• Applications: Flow cytometry, immunoprecipitation, quantitative Western blotting

Polyclonal Antibodies (e.g., 28351-1-AP, 20845-1-AP)

• Advantages: Recognition of multiple epitopes, higher sensitivity, robust signal

• Best for: Initial protein characterization, detection of denatured proteins

• Applications: Immunohistochemistry, initial Western blot screening

For critical applications requiring reproducibility across multiple experiments, monoclonal antibodies may offer advantages, while polyclonals may provide better signal in challenging applications .

What is the recommended protocol for PRKAR2B co-immunoprecipitation experiments?

For successful co-immunoprecipitation of PRKAR2B interacting proteins:

• Lysate preparation:

  • Harvest cells/tissues in non-denaturing lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate)

  • Include protease and phosphatase inhibitors

  • Clarify by centrifugation at 14,000g for 10 minutes at 4°C

• Immunoprecipitation:

  • Use 0.5-4.0 μg antibody per 1.0-3.0 mg protein lysate

  • Incubate with lysate overnight at 4°C with gentle rotation

  • Add protein A/G beads for 1-2 hours

  • Wash 4-5 times with lysis buffer

• Elution and analysis:

  • Elute with SDS sample buffer at 95°C for 5 minutes

  • Analyze by SDS-PAGE followed by Western blotting

This protocol has been successfully used to immunoprecipitate PRKAR2B from mouse brain tissue and human brain lysates .

How can I optimize PRKAR2B detection in neuronal samples?

For improved detection in neuronal tissues and cells:

• Sample preparation:

  • For brain tissue: Use specialized neural tissue lysis buffers containing 1% SDS

  • For cultured neurons: Direct lysis in 2X SDS sample buffer may improve yield

• Application-specific recommendations:

  • Western blot: Use 1:500-1:2000 dilution with overnight incubation at 4°C

  • IHC/IF: Extended antigen retrieval (20-30 minutes) may improve signal

  • IF in cultured neurons: Fix with 4% PFA for 10-15 minutes, permeabilize with 0.1-0.3% Triton X-100

• Validated positive controls:

  • Mouse/rat brain tissue (particularly cerebellum)

  • Neuro-2a cells

  • SH-SY5Y cells

These optimizations have been validated for detection of PRKAR2B in various neuronal models .

How is PRKAR2B being studied in relation to signal transduction pathways?

Current research focuses on PRKAR2B's role in:

• cAMP signaling: As a regulatory subunit of PKA, PRKAR2B controls cAMP-dependent phosphorylation cascades

• Membrane association: Type II regulatory chains mediate membrane localization through anchoring proteins, including MAP2 kinase

• CREB1 interaction: PRKAR2B has been shown to interact with and suppress CREB1 transcriptional activity in activated T cells

• Energy metabolism: Knockout studies suggest PRKAR2B regulates energy balance and adiposity

• Gene induction: PRKAR2B may mediate gene expression induced by certain pharmaceuticals like haloperidol

These pathways represent opportunities for developing targeted approaches to modulate PRKAR2B activity in various physiological and pathological contexts .

What are the current challenges in studying PRKAR2B post-translational modifications?

Researchers face several challenges when investigating PRKAR2B modifications:

• Phosphorylation site specificity: Multiple phosphorylation sites exist, with Ser113 being particularly important

• Antibody selection: Phospho-specific antibodies require careful validation

• Sample preparation: Phosphatase inhibitors are crucial to preserve phosphorylation status

• Technical considerations:

  • Use freshly prepared samples to minimize dephosphorylation

  • Include positive controls with known phosphorylation status

  • Consider lambda phosphatase treatment as a negative control

Phospho-specific antibodies targeting sites like Ser113 are now available and can help differentiate the activity state of PRKAR2B in experimental systems .