PRKAR1B Antibody

What is PRKAR1B and what cellular functions does it regulate?

PRKAR1B is the gene encoding the R1β subunit of cyclic AMP-dependent protein kinase A (PKA). It functions as a regulatory subunit involved in cAMP signaling pathways in cells . The protein plays a crucial role in various physiological processes, including neurodevelopment. Recent studies have identified PRKAR1B as significantly associated with neurodevelopmental disorders, with enrichment of de novo missense variants found in large patient cohorts with intellectual disability and autism spectrum disorder .

The functional importance of PRKAR1B has been demonstrated in vitro, where variant-harboring PRKAR1B expression constructs showed altered basal PKA enzymatic activity. Specifically, lysates of cells transfected with three different variant-harboring constructs (p.Glu196Lys, p.Gln167Leu, and p.Arg335Trp) revealed significantly decreased basal PKA enzymatic activity compared to wild-type constructs .

What applications are PRKAR1B antibodies suitable for in research settings?

PRKAR1B antibodies are validated for multiple research applications, with specific recommendations depending on the antibody type (polyclonal vs. monoclonal) and host species. Common applications include:

It's important to note that optimal dilutions may be sample-dependent, and researchers should conduct titration experiments to determine ideal conditions for their specific experimental systems .

How should I optimize Western blot protocols for PRKAR1B detection?

When optimizing Western blot protocols for PRKAR1B detection, consider the following methodological approach:

• Sample preparation: PRKAR1B has been successfully detected in mouse brain tissue, transfected HEK-293 cells, and HeLa cells . Use appropriate lysis buffers containing protease inhibitors to preserve protein integrity.

• Expected molecular weight: The calculated molecular weight of PRKAR1B is 43 kDa (381 amino acids), but the observed molecular weight typically ranges between 45-50 kDa . This discrepancy should be considered when interpreting results.

• Antibody selection: Both polyclonal (e.g., rabbit) and monoclonal (e.g., mouse) antibodies are available. Polyclonal antibodies like 17991-1-AP have been validated for detecting human, mouse, and rat PRKAR1B .

• Blocking and dilution: Use a standard blocking solution (5% non-fat milk or BSA) and dilute primary antibodies according to manufacturer recommendations (typically 1:500-1:2000 for PRKAR1B antibodies) .

• Detection: Standard HRP-conjugated secondary antibodies compatible with your primary antibody host species are recommended, followed by ECL-based detection systems.

For challenging samples or to improve specificity, consider antigen retrieval methods similar to those used in IHC protocols, which include TE buffer at pH 9.0 or citrate buffer at pH 6.0 .

What are the recommended conditions for immunohistochemistry using PRKAR1B antibodies?

For optimal immunohistochemistry results using PRKAR1B antibodies, follow these methodological guidelines:

• Tissue preparation: PRKAR1B antibodies have been validated on human breast cancer tissue and mouse brain tissue sections . Standard formalin fixation and paraffin embedding protocols are appropriate.

• Antigen retrieval: This is a critical step for PRKAR1B detection. The recommended method is:

  • Primary option: TE buffer at pH 9.0

  • Alternative option: Citrate buffer at pH 6.0

• Antibody dilution: For IHC applications, use PRKAR1B antibodies at dilutions ranging from 1:50 to 1:500, optimizing for your specific tissue sample and antibody .

• Detection system: Standard biotin-streptavidin or polymer-based detection systems are compatible with PRKAR1B antibodies.

• Controls: Always include positive controls (such as brain tissue) and negative controls (primary antibody omission) to validate staining specificity.

For researchers investigating neurodevelopmental disorders, brain tissue sections are particularly relevant since PRKAR1B expression has been demonstrated during human embryonal development .

How can PRKAR1B antibodies be used to study its role in neurodevelopmental disorders?

PRKAR1B has emerged as a significant candidate gene in neurodevelopmental disorders (NDDs), with strong evidence supporting its involvement in autism spectrum disorder (ASD) and intellectual disability . Researchers can use PRKAR1B antibodies to investigate this connection through several methodological approaches:

• Expression analysis in patient-derived samples: Using validated PRKAR1B antibodies for Western blot or IHC to compare expression levels and patterns between neurotypical controls and NDD patient samples. Focus on brain regions implicated in ASD and intellectual disability.

• Functional studies of variant effects: Recent research identified several pathogenic variants, particularly c.1003C>T (p.Arg335Trp), which was found in four unrelated individuals . Researchers can generate these variants in expression constructs and use PRKAR1B antibodies to:

  • Assess protein stability and expression levels

  • Determine subcellular localization changes

  • Evaluate protein-protein interactions

• Developmental expression mapping: PRKAR1B expression during embryonic brain development can be tracked using immunohistochemistry with PRKAR1B antibodies, providing insights into temporal and spatial expression patterns relevant to neurodevelopmental processes .

• Correlation with phenotypes: Clinical phenotypes associated with PRKAR1B variants include global developmental delay, autism spectrum disorder, apraxia/dyspraxia, and reduced pain sensitivity in some cases . Researchers can use PRKAR1B antibodies to investigate expression in relevant neural circuits.

This approach has already yielded valuable insights, as demonstrated by in vitro analyses that revealed altered basal PKA activity in cells transfected with variant-harboring PRKAR1B expression constructs .

What are the key considerations when interpreting PRKAR1B antibody signals in the context of variant studies?

When using PRKAR1B antibodies to study the effects of genetic variants, researchers should consider these methodological aspects for accurate data interpretation:

• Variant localization and functional domains: The identified pathogenic variants (p.Glu196Lys, p.Gln167Leu, and p.Arg335Trp) are situated within annotated nucleotide binding regions according to UniProt database . Antibodies targeting different epitopes may have varying sensitivity to conformational changes induced by these variants.

• Epitope accessibility: When selecting a PRKAR1B antibody, consider the epitope location relative to the variant being studied:

  • Antibodies targeting AA 1-90 may be suitable for N-terminal variants

  • Antibodies recognizing AA 190-290 target the regulatory subunit region

  • Full-length antibodies may provide comprehensive detection regardless of variant location

• Specificity validation: Cross-reactivity should be carefully assessed, particularly when studying PRKAR1B in different species. Available antibodies have varying reactivity patterns across human, mouse, and rat samples .

• Functional impact assessment: When studying variants like p.Arg335Trp, which showed decreased basal PKA enzymatic activity in vitro , consider combining antibody-based detection methods with functional assays to correlate expression/localization with enzyme activity.

• Data normalization: Always compare variant-containing samples with wild-type controls processed identically, and use loading controls appropriate for the subcellular compartment where PRKAR1B is expected to localize.

This careful interpretation approach is essential given that different variants may affect protein function through distinct mechanisms, as demonstrated by the observation that while basal PKA activity was decreased with all three variants studied, total or cAMP-stimulated PKA activity showed more variable effects .

How can dual immunofluorescence with PRKAR1B antibodies be optimized for co-localization studies?

For researchers investigating PRKAR1B interactions or subcellular localization through co-immunofluorescence, consider these methodological recommendations:

• Compatible antibody selection: When designing co-localization experiments, select PRKAR1B antibodies from different host species than your second target protein antibody. Available options include:

  • Mouse monoclonal PRKAR1B antibodies (clones 1F8, 2A3)

  • Rabbit polyclonal PRKAR1B antibodies

• Fixation optimization: For preserving PRKAR1B epitopes while maintaining cellular architecture:

  • For membrane-associated studies: 4% paraformaldehyde (10-15 minutes)

  • For nuclear studies: methanol:acetone (1:1) fixation (10 minutes at -20°C)

• Blocking parameters: To minimize non-specific binding:

  • Standard: 5% normal serum from the species of your secondary antibody

  • Alternative for high background: 2% BSA with 0.1% Triton X-100

• Sequential versus simultaneous incubation:

  • For antibodies from different species: simultaneous incubation is possible

  • For same-species antibodies: sequential detection with direct conjugates or Fab fragment blocking between steps

• Control experiments:

  • Single primary antibody controls with both secondary antibodies to check cross-reactivity

  • Secondary-only controls to assess non-specific binding

  • Known pattern controls to verify expected PRKAR1B localization

This approach is particularly valuable for investigating PRKAR1B interaction with other proteins in signaling pathways, especially those related to c-Jun N-terminal kinase and mitogen-activated protein kinase cascades, which have been implicated in neurodevelopmental disorders .

What methods can be employed to study PKA activity alterations in the context of PRKAR1B variants?

Investigating PKA enzymatic activity changes caused by PRKAR1B variants requires specialized approaches that can complement antibody-based detection. Based on successful methodologies from published research , consider these protocols:

• Expression construct generation:

  • Create expression vectors containing wild-type PRKAR1B and variant sequences (e.g., p.Glu196Lys, p.Gln167Leu, p.Arg335Trp)

  • Tag with fluorescent markers (e.g., pVenus) for expression verification

• Transfection optimization:

  • Use HEK293 cells as they have been validated for PRKAR1B expression studies

  • Optimize transfection conditions to achieve consistent expression levels

  • Confirm expression using PRKAR1B antibodies via Western blot or immunofluorescence

• PKA activity measurement:

  • Basal activity: Assess phosphorylation of PKA substrates in unstimulated conditions

  • cAMP-stimulated activity: Treat with cAMP analogs or adenylyl cyclase activators

  • Use commercially available PKA activity assay kits based on phosphorylation of specific substrates

• Data analysis considerations:

  • Statistical approach: ANOVA for comparing multiple variants as demonstrated in previous research

  • Normalization: Account for transfection efficiency and expression level differences

  • Controls: Include untransfected cells and cells transfected with empty vectors

This combined approach has successfully demonstrated that cells transfected with variant-harboring constructs (p.Glu196Lys, p.Gln167Leu, and p.Arg335Trp) exhibited significantly decreased basal PKA enzymatic activity compared to wild-type (ANOVA, p = 0.0012) .

How should researchers approach species cross-reactivity when using PRKAR1B antibodies?

PRKAR1B antibodies show varying cross-reactivity across species, which is an important consideration for comparative studies or when selecting animal models. Follow these methodological guidelines:

• Antibody selection based on documented cross-reactivity:

  • Some antibodies are human-specific (e.g., certain monoclonal antibodies)

  • Others react with human, mouse, and rat samples (e.g., rabbit polyclonal 17991-1-AP)

  • Review full validation data before selecting an antibody for cross-species applications

• Sequence homology analysis:

  • Prior to experimentation, compare PRKAR1B sequences across target species

  • Focus on the epitope region recognized by your selected antibody

  • Higher homology in the epitope region predicts better cross-reactivity

• Validation strategies for untested species:

  • Positive control: Include samples from validated species alongside your test species

  • Peptide competition: Pre-incubate antibody with immunizing peptide to confirm specificity

  • Multiple antibody approach: Use antibodies targeting different epitopes to confirm results

• Optimization for specific applications:

  • Western blot: May require species-specific adjustments to lysis buffers and blocking agents

  • IHC/IF: May need modified fixation and antigen retrieval protocols for different species

  • Consider species-specific secondary antibodies to minimize background

This thoughtful approach to cross-reactivity is particularly important for translational research, as PRKAR1B has been implicated in both human neurodevelopmental disorders and studied in various model organisms to understand its biological functions .

What are common issues in Western blot detection of PRKAR1B and how can they be resolved?

Researchers may encounter several challenges when detecting PRKAR1B via Western blotting. Here are methodological solutions to common problems:

• Incorrect band size:

  • Expected size: Calculated molecular weight is 43 kDa, but observed range is typically 45-50 kDa

  • Solution: Ensure appropriate ladder selection and consider post-translational modifications that may affect migration

  • Validation: Use positive control lysates (mouse brain tissue, HEK-293 cells, or HeLa cells) for size comparison

• Weak or absent signal:

  • Primary cause: Insufficient protein, inadequate transfer, or antibody concentration issues

  • Solution: Increase protein loading (30-50 μg recommended), optimize transfer conditions for proteins >40 kDa, and adjust antibody concentration (try 1:500 dilution initially)

  • Alternative: Consider more sensitive detection systems like ECL-Plus or fluorescent secondary antibodies

• High background:

  • Causes: Insufficient blocking, antibody concentration too high, or inadequate washing

  • Solution: Extend blocking time (2 hours at room temperature or overnight at 4°C), dilute antibody further, and increase wash duration/frequency

  • Buffer modification: Add 0.1% Tween-20 to wash buffer and consider 5% BSA instead of milk for blocking

• Multiple bands:

  • Explanation: Could indicate degradation products, splice variants, or cross-reactivity

  • Verification: Compare pattern with literature reports and positive controls

  • Resolution: Try different lysis conditions with complete protease inhibitor cocktails

These troubleshooting approaches are based on validated protocols for PRKAR1B antibodies and general principles of Western blot optimization tailored to this specific protein's characteristics .

How can non-specific staining be minimized in PRKAR1B immunohistochemistry?

Non-specific staining is a common challenge in PRKAR1B immunohistochemistry. Implement these methodological strategies to improve signal specificity:

• Optimized antigen retrieval:

  • Primary recommendation: TE buffer at pH 9.0

  • Alternative approach: Citrate buffer at pH 6.0

  • Timing: 15-20 minutes at sub-boiling temperatures, followed by 20-minute cooling

• Endogenous enzyme blocking:

  • For peroxidase-based detection: 3% hydrogen peroxide in methanol (10 minutes)

  • For alkaline phosphatase: Levamisole (2mM in detection solution)

  • For both: Commercial dual-block solutions are available

• Background reduction techniques:

  • Pre-incubation with serum from secondary antibody species (5%, 30 minutes)

  • Add 0.1-0.3% Triton X-100 to reduce hydrophobic interactions

  • Include 0.1% BSA in antibody diluent to stabilize specific binding

• Antibody optimization:

  • Start with manufacturer's recommended dilution range (1:50-1:500)

  • Perform titration experiments to determine optimal concentration

  • Consider overnight incubation at 4°C instead of shorter incubations at room temperature

• Additional controls:

  • Absorption control: Pre-incubate primary antibody with recombinant PRKAR1B

  • Isotype control: Use non-immune IgG matching primary antibody host/isotype

  • Tissue negative control: Include tissues known not to express PRKAR1B

These approaches have been validated for PRKAR1B detection in human breast cancer tissue and mouse brain tissue, which serve as positive controls for optimization experiments .

How can PRKAR1B antibodies contribute to understanding neurodegenerative conditions?

Beyond neurodevelopmental disorders, PRKAR1B has been associated with rare hereditary neurodegenerative conditions characterized by R1β-positive inclusions in affected neurons . Researchers can employ PRKAR1B antibodies to investigate these connections through several methodological approaches:

• Pathological inclusion characterization:

  • Immunohistochemistry using PRKAR1B antibodies on post-mortem brain tissues

  • Co-labeling with neurodegeneration markers (tau, α-synuclein, TDP-43)

  • Analysis of inclusion morphology, distribution, and correlation with clinical features

• In vitro aggregation studies:

  • Using PRKAR1B antibodies to monitor aggregation propensity of wild-type versus variant proteins

  • Time-course experiments to track inclusion formation

  • Co-immunoprecipitation to identify interacting partners in aggregates

• Animal model development and validation:

  • PRKAR1B antibodies can verify transgene expression in genetically modified animals

  • Track age-dependent accumulation of R1β-positive inclusions

  • Correlate inclusion formation with behavioral and physiological phenotypes

• Biomarker potential exploration:

  • Investigate whether PRKAR1B or its fragments can be detected in CSF or blood

  • Compare levels between neurodegenerative patients and controls

  • Develop sensitive immunoassays for early detection

This research direction represents an important intersection between neurodevelopmental and neurodegenerative research, potentially revealing common pathways or mechanisms that could inform therapeutic approaches for both condition types .

What methodological approaches can be used to study PRKAR1B in the context of pain sensitivity phenotypes?

The observation that reduced pain sensitivity was reported in three individuals with the c.1003C>T (p.Arg335Trp) PRKAR1B variant opens an intriguing research direction. Researchers can employ these methodological approaches:

• Tissue-specific expression analysis:

  • Use PRKAR1B antibodies for immunohistochemistry on dorsal root ganglia, spinal cord, and brain regions involved in pain processing

  • Compare expression patterns between wild-type and variant models

  • Quantify co-localization with known pain pathway markers (substance P, CGRP, etc.)

• Functional studies in sensory neurons:

  • Transfect primary sensory neurons with wild-type or variant PRKAR1B constructs

  • Use PRKAR1B antibodies to confirm expression and localization

  • Assess changes in:

    • Calcium signaling responses to nociceptive stimuli

    • Electrophysiological properties

    • Neurite outgrowth and morphology

• Signaling pathway investigation:

  • Examine phosphorylation status of known PKA substrates in pain pathways

  • Assess cAMP response element-binding protein (CREB) activation

  • Investigate interaction with other pain-related signaling molecules

• Animal model phenotyping:

  • Generate knock-in models expressing the p.Arg335Trp variant

  • Use PRKAR1B antibodies to confirm expression and localization

  • Conduct comprehensive pain behavioral testing (thermal, mechanical, chemical stimuli)

  • Correlate behavioral phenotypes with molecular alterations

This line of investigation could provide valuable insights into both the mechanisms of PRKAR1B-related disorders and potentially novel pain pathway biology, which might ultimately inform analgesic drug development strategies .

What future directions in PRKAR1B antibody development would benefit the research community?

As research on PRKAR1B continues to expand, particularly in relation to neurodevelopmental disorders and neurodegenerative conditions, several developments in antibody technology would significantly advance the field:

• Variant-specific antibodies:

  • Development of antibodies specifically recognizing common pathogenic variants (particularly p.Arg335Trp)

  • These would enable direct detection of mutant proteins in patient samples without requiring genetic testing

  • Applications would include screening biobank samples to identify carriers and studying variant-specific effects

• Phosphorylation-state specific antibodies:

  • Antibodies recognizing specific phosphorylation states of PRKAR1B

  • These would allow monitoring of PRKAR1B regulatory status in different physiological and pathological conditions

  • Particularly useful for studying dynamic signaling changes in response to cAMP

• Humanized antibodies for therapeutic potential:

  • If PRKAR1B misfolding contributes to pathology, humanized antibodies could be developed for therapeutic applications

  • These could potentially target misfolded proteins or aggregates for clearance

  • May represent a novel approach for conditions with PRKAR1B-positive inclusions

• Multifunctional probe development:

  • Antibody-based imaging probes combining PRKAR1B recognition with functional readouts

  • Examples include FRET-based sensors to monitor PRKAR1B-protein interactions in live cells

  • These would enable dynamic studies of PRKAR1B function in real-time