PRKACA Antibody, Biotin conjugated

What is PRKACA and why is it significant in biomedical research?

PRKACA (protein kinase, cAMP-dependent, catalytic, alpha) is a key catalytic subunit of protein kinase A with a molecular weight of approximately 40.6 kDa (calculated) and 38-43 kDa (observed in experiments). This protein is widely expressed across multiple tissue types and functions as a member of the AGC Ser/Thr protein kinase family . PRKACA plays crucial roles in GPCR signaling pathways and carbohydrate metabolism and homeostasis, making it a significant target in various research fields .

The protein localizes to multiple cellular compartments including the cell membrane, nucleus, cytoplasmic vesicles, mitochondria, and cytoplasm . Its involvement in numerous signaling pathways makes it relevant to research in oncology, metabolism, and cell signaling. Particularly notable is the DNAJB1-PRKACA fusion protein, which serves as an oncogenic driver in fibrolamellar hepatocellular carcinoma, a lethal disease with limited therapeutic options .

What sample types and experimental conditions are optimal for PRKACA antibody applications?

PRKACA antibodies have demonstrated reactivity with samples from multiple species including human, mouse, and rat tissues and cell lines . The following table summarizes optimal sample types based on validated experimental data:

For IHC applications, antigen retrieval can be performed using TE buffer at pH 9.0 or alternatively with citrate buffer at pH 6.0 . The high conservation of PRKACA across species enables cross-reactivity, providing flexibility in experimental design across different model systems.

How should researchers validate PRKACA antibody specificity before experimental use?

Validating antibody specificity is essential for reliable experimental outcomes. For PRKACA antibodies, consider the following validation approaches:

• Positive and negative controls: Use cell lines with known PRKACA expression levels. Based on published data, HeLa, Neuro-2a, and SH-SY5Y cells serve as reliable positive controls .

• Molecular weight verification: Confirm that the detected band falls within the expected range of 38-43 kDa on Western blots .

• Knockdown/knockout validation: Verify specificity by comparing detection between wildtype and PRKACA-depleted samples. Published literature includes knockout validation for certain PRKACA antibodies .

• Cross-verification: Compare results using multiple antibodies targeting different epitopes of PRKACA to ensure consistent detection patterns.

• Blocking peptide competition: Pre-incubate the antibody with the immunogen peptide to confirm specificity through signal reduction.

The antibody's specificity can be further confirmed by comparing experimental results with published data where PRKACA has been detected in similar experimental conditions.

What are the optimal conditions for detecting PRKACA in multiplex immunoassays?

When incorporating PRKACA antibodies into multiplex immunoassays, several technical considerations should be addressed:

• Antibody compatibility: Select antibodies raised in different host species to avoid cross-reactivity in multiple labeling experiments. PRKACA antibodies are available in rabbit host format, which should be paired with antibodies from other species .

• Signal optimization: For fluorescence-based detection methods, consider the following parameters:

  • Fixation: 4% paraformaldehyde for 15-20 minutes at room temperature

  • Permeabilization: 0.1-0.3% Triton X-100 for 5-10 minutes

  • Blocking: 5% normal serum (matched to secondary antibody host) with 1% BSA for 30-60 minutes

• Detection strategy: For biotin-conjugated antibodies, use streptavidin coupled to a fluorophore with minimal spectral overlap with other detection reagents. Optimal dilution ranges for biotin-conjugated antibodies typically require titration starting at 1:200-1:500.

• Controls: Include single-stained controls to assess bleed-through and secondary-only controls to evaluate background signal.

• Sequential staining: Consider sequential rather than simultaneous staining protocols when using antibodies with similar properties to minimize cross-reactivity.

When optimizing multiplex protocols, each antibody should be individually validated before combining into the multiplex system to ensure specificity is maintained in the more complex environment.

How can researchers troubleshoot inconsistent PRKACA antibody performance across different experimental systems?

Inconsistent antibody performance can arise from multiple factors. The following troubleshooting guide addresses common issues specifically relevant to PRKACA antibody applications:

• Sample preparation variations:

  • For Western blot: Ensure complete protein denaturation using sufficient SDS and heating (95-100°C for 5 minutes)

  • For IHC/IF: Optimize fixation (4% PFA or 10% formalin) and antigen retrieval methods (TE buffer pH 9.0 or citrate buffer pH 6.0)

• Epitope accessibility issues:

  • PRKACA has multiple subcellular localizations (membrane, nucleus, cytoplasm, mitochondria)

  • Different fixation and permeabilization protocols may be required depending on the targeted subcellular compartment

  • For membrane-associated PRKACA, mild detergents like 0.1% Tween-20 may be preferable to stronger agents

• Antibody dilution optimization:

  • Perform systematic dilution series specific to each application:

    • WB: 1:500-1:6000

    • IHC: 1:50-1:500

    • IF/ICC: 1:50-1:500

• Post-translational modifications:

  • PRKACA function is regulated by phosphorylation

  • Phosphorylation-dependent epitope masking may affect antibody recognition

  • Consider phosphatase treatment of samples if epitope recognition is influenced by phosphorylation status

• Lot-to-lot variations:

  • Document lot numbers and maintain reference samples for comparison when switching antibody lots

  • Request certificate of analysis with batch-specific validation data from suppliers

For biotin-conjugated antibodies specifically, additional considerations include streptavidin blocking steps for endogenous biotin and optimizing streptavidin-conjugate concentrations to minimize background while maintaining sensitivity.

What are the critical considerations when studying the DNAJB1-PRKACA fusion protein in fibrolamellar hepatocellular carcinoma?

The DNAJB1-PRKACA fusion protein represents a unique oncogenic driver in fibrolamellar hepatocellular carcinoma (FL-HCC) . When studying this fusion protein, researchers should consider:

• Antibody epitope location:

  • Select antibodies targeting the PRKACA portion that is preserved in the fusion protein

  • Differential detection between wild-type PRKACA and the fusion protein may require antibodies targeting the fusion junction

• Expression system considerations:

  • The fusion protein maintains kinase activity but with altered substrate specificity

  • Expression levels may differ from wild-type PRKACA

  • Control experiments should include both wild-type PRKACA and fusion protein-expressing systems

• Immunological significance:

  • The fusion junction creates unique neoepitopes that can be immunogenic

  • These neoepitopes are processed and presented on HLA class I and II molecules

  • For immunotherapeutic applications, both CD8+ and CD4+ T cell responses against the fusion protein have been documented

• Technical validation approaches:

  • Verify fusion protein expression using paired antibodies against both DNAJB1 and PRKACA portions

  • RNA-seq or RT-PCR validation of the fusion transcript alongside protein detection

  • Mass spectrometry-based confirmation of the fusion junction peptides

The DNAJB1-PRKACA fusion has been successfully targeted with peptide-based immunotherapy, highlighting its importance as both a diagnostic marker and therapeutic target in FL-HCC research .

How does biotin conjugation affect experimental design and detection sensitivity for PRKACA antibodies?

Biotin conjugation provides specific advantages and requires methodological adjustments when working with PRKACA antibodies:

• Signal amplification considerations:

  • Biotin-streptavidin interaction provides strong (Kd ~10^-15 M) and stable binding

  • Multiple biotin molecules per antibody can increase detection sensitivity

  • Optimal working dilutions for biotinylated antibodies may differ from unconjugated versions, typically requiring 2-3 fold higher dilutions

• Endogenous biotin interference:

  • Certain tissues (liver, kidney, brain) contain high levels of endogenous biotin

  • Incorporate avidin/streptavidin blocking steps (10-15 minutes with commercial blocking kit) before applying biotinylated antibodies

  • Consider the use of specialized blocking reagents (e.g., streptavidin/biotin blocking kit) for tissues with high endogenous biotin

• Detection system selection:

  • Enzymatic detection: Streptavidin-HRP or streptavidin-AP followed by appropriate substrate

  • Fluorescent detection: Streptavidin conjugated to fluorophores (Alexa 488, 555, 647)

  • Choosing detection systems with minimal background in the tissue/cells of interest

• Storage considerations:

  • Biotin-conjugated antibodies typically maintain stability at -20°C with 50% glycerol

  • Avoid repeated freeze-thaw cycles which can degrade both the antibody and biotin moiety

  • Prepare single-use aliquots when possible

• Multiplexing advantages:

  • Biotin-streptavidin pairing enables flexible secondary detection systems

  • Compatible with TSA (tyramide signal amplification) for ultra-sensitive detection

  • Can be combined with directly labeled antibodies in multiplex experiments

When directly comparing results between biotinylated and non-biotinylated PRKACA antibodies, include appropriate controls to account for potential differences in sensitivity and background.

What are the optimal protocols for detecting PRKACA in challenging sample types?

Detecting PRKACA in challenging samples requires optimized protocols tailored to specific sample characteristics:

• Formalin-fixed paraffin-embedded (FFPE) tissues:

  • Extended antigen retrieval: 20-30 minutes in TE buffer pH 9.0 at 95-100°C

  • Signal amplification: Consider tyramide signal amplification for low-abundance detection

  • Background reduction: Include additional blocking steps (0.3% hydrogen peroxide, avidin/biotin blocking)

  • Optimal dilution range: 1:50-1:200 (more concentrated than fresh samples)

• Brain tissues with high lipid content:

  • Extended fixation: 24-48 hours in 4% PFA

  • Permeabilization enhancement: Add 0.1-0.3% Triton X-100 to all antibody diluents

  • Background reduction: Include 0.1-0.3 M glycine to reduce autofluorescence

  • Consider antigen retrieval even for frozen sections to improve epitope accessibility

• Archived/degraded samples:

  • Target selection: Focus on stable epitopes less affected by long-term storage

  • Signal recovery: Apply heat-induced epitope retrieval with pH optimization (test both acidic and basic buffers)

  • Alternative detection: Consider proximity ligation assay for improved specificity in partially degraded samples

• Samples with high background:

  • For tissues with high endogenous peroxidase: Extended hydrogen peroxide blocking (0.3-3% for 15-30 minutes)

  • For highly autofluorescent tissues: Pretreatment with Sudan Black B (0.1-0.3%) or commercial autofluorescence quenchers

  • For tissues with high IgG content: Include mouse/rabbit IgG blocking steps before primary antibody application

• Low abundance detection:

  • Consider alternative lysis buffers containing phosphatase inhibitors if phosphorylated forms are of interest

  • Employ protein concentration techniques (immunoprecipitation with 0.5-4.0 μg antibody per 1-3 mg lysate)

  • Consider enzymatic signal amplification methods (catalyzed reporter deposition)

These specialized protocols should be validated on control samples before application to experimental samples of high value or limited availability.

How should researchers design experiments to study PRKACA interactions with binding partners and signaling pathways?

Investigating PRKACA protein interactions and signaling requires specialized experimental approaches:

• Co-immunoprecipitation protocols:

  • Lysis buffer optimization: Use non-denaturing buffers containing 1% NP-40 or 0.5% Triton X-100 with protease inhibitors

  • Antibody selection: Use antibodies validated for IP (0.5-4.0 μg per reaction)

  • Capture approach: Protein A/G beads or directly conjugated magnetic beads

  • Controls: Include IgG control immunoprecipitations and input lysate lanes

  • Detection: Blot for both PRKACA and suspected interaction partners

• Proximity ligation assay (PLA) for in situ interaction studies:

  • Cell preparation: 4% PFA fixation for 15 minutes, 0.2% Triton X-100 permeabilization

  • Antibody selection: Combine PRKACA antibody with antibodies against suspected interaction partners (from different host species)

  • Detection: Follow commercial PLA protocols with optimization of antibody concentrations

  • Controls: Include single primary antibody controls and known interaction pairs

• Functional interaction assays:

  • Kinase activity assays: Measure PRKACA activity using phospho-specific antibodies against known substrates

  • Inhibitor studies: Compare phosphorylation patterns with and without selective PKA inhibitors

  • Subcellular fractionation: Track compartment-specific interactions through biochemical separation followed by immunoblotting

  • FRET/BRET approaches: For dynamic, live-cell monitoring of protein-protein interactions

• Mass spectrometry-based interactome analysis:

  • Immunoprecipitate PRKACA using validated antibodies (optimally 2-5 μg per IP)

  • Process samples for LC-MS/MS analysis

  • Employ appropriate controls (IgG IPs, competing peptides)

  • Validate key interactions through orthogonal methods (co-IP, PLA)

When studying the DNAJB1-PRKACA fusion protein specifically, consider comparative interaction analyses between wild-type PRKACA and the fusion protein to identify altered protein interactions that may contribute to oncogenic activity .

How should researchers interpret discrepancies between PRKACA antibody results and transcriptomic data?

Discrepancies between protein detection and gene expression data are common in biological research. For PRKACA specifically:

• Potential sources of discrepancy:

  • Post-transcriptional regulation: miRNA targeting of PRKACA mRNA

  • Post-translational modifications affecting epitope recognition

  • Protein stability differences across tissue types

  • Subcellular localization changes affecting extraction efficiency

  • Presence of multiple isoforms (up to 2 different isoforms have been reported)

• Validation approaches:

  • Multi-antibody verification: Use antibodies targeting different PRKACA epitopes

  • Orthogonal methods: Combine Western blot with targeted mass spectrometry

  • Subcellular fractionation: Assess compartment-specific distribution

  • Translation inhibition studies: Evaluate protein half-life using cycloheximide chase

• Data integration frameworks:

  • Normalize protein data to housekeeping proteins and mRNA data to reference genes

  • Consider time-course studies to capture potential delays between transcription and translation

  • Employ statistical methods designed for multi-omics data integration

• Biological interpretation:

  • PRKACA function is heavily regulated post-translationally

  • Protein activity may not correlate directly with abundance

  • Consider activity-based assays alongside abundance measurements

For the most comprehensive understanding, researchers should combine protein detection methods with activity measurements and localization studies, particularly when transcriptomic and proteomic data appear contradictory.

What are the key considerations when using PRKACA antibodies in immunotherapeutic research applications?

PRKACA has emerging importance in immunotherapy research, particularly through the DNAJB1-PRKACA fusion in FL-HCC . When using PRKACA antibodies in this context, consider:

• Epitope selection for therapeutic targeting:

  • The DNAJB1-PRKACA fusion creates unique junction epitopes recognized by both CD8+ and CD4+ T cells

  • These fusion-specific epitopes are processed and presented on HLA class I and HLA class II molecules

  • Antibodies targeting these junction epitopes would be highly specific for the oncogenic fusion

• Validation for immunotherapy research:

  • Confirm cellular processing and HLA-restricted presentation through mass spectrometry-based immunopeptidome analysis

  • Validate epitope immunogenicity through T cell activation assays

  • Consider HLA type variations when designing epitope-targeted approaches

• Applications in monitoring therapeutic responses:

  • Track changes in PRKACA or fusion protein expression during treatment

  • Monitor neoantigen-specific T cell responses using matched antibody-based detection systems

  • Assess tumor infiltrating lymphocytes in relation to PRKACA/fusion protein expression

• Translational considerations:

  • DNAJB1-PRKACA fusion-derived peptide vaccines have shown promise in inducing multifunctional CD4+ T cells with an activated T-helper 1 phenotype

  • Combining antibody-based detection with T cell receptor sequencing can provide comprehensive immune response monitoring

  • Consider combinatorial approaches targeting both the fusion protein and the tumor microenvironment

For immunotherapy applications, the specificity of antibodies for distinguishing between wild-type PRKACA and the fusion protein is particularly critical for accurate patient stratification and response monitoring.

What emerging technologies are enhancing PRKACA antibody applications in cutting-edge research?

Emerging technologies are expanding the utility of PRKACA antibodies in several innovative directions:

• Single-cell proteomics applications:

  • Integration with mass cytometry (CyTOF) for high-dimensional analysis

  • Adaptation to microfluidic-based single-cell Western blotting

  • Incorporation into spatial proteomics platforms for tissue-level analysis

  • Development of photocleavable antibody-DNA conjugates for single-cell sequencing

• Live-cell imaging innovations:

  • Development of conformation-sensitive antibody derivatives

  • Integration with optogenetic systems for activity-dependent detection

  • Nanobody-based approaches for improved intracellular penetration

  • FRET-based biosensors incorporating antibody-derived binding domains

• Therapeutic antibody development potential:

  • Engineered antibodies recognizing the DNAJB1-PRKACA fusion junction

  • Antibody-drug conjugates specifically targeting fusion-positive cells

  • Bispecific antibodies linking T cells to fusion-expressing cancer cells

  • CAR-T cell development based on antibody-derived binding domains

• Structural biology applications:

  • Antibody-assisted cryo-EM for capturing specific PRKACA conformational states

  • Antibody-mediated crystallization of challenging protein complexes

  • In-cell structural studies combining antibody detection with proximity labeling

These technological innovations are poised to transform both basic research into PRKACA biology and translational applications targeting PRKACA-related pathologies, particularly in the context of the DNAJB1-PRKACA fusion protein's role in fibrolamellar hepatocellular carcinoma .

How can researchers contribute to improved standardization of PRKACA antibody applications?

Standardization efforts are critical for enhancing reproducibility in PRKACA antibody applications:

• Validation reporting standards:

  • Document complete validation protocols including positive and negative controls

  • Report antibody catalog numbers, lot numbers, and dilutions used

  • Include details on sample preparation, blocking, and detection methods

  • Share raw unprocessed images alongside processed data

• Cross-laboratory validation initiatives:

  • Participate in multi-laboratory validation studies

  • Share standard operating procedures through repositories

  • Contribute to antibody validation databases with application-specific data

  • Develop reference standards for PRKACA detection across applications

• New technology implementation:

  • Adopt orthogonal validation methods (mass spectrometry, CRISPR controls)

  • Implement quantitative metrics for antibody performance

  • Explore automation to reduce technical variability

  • Adopt digital pathology tools for standardized image analysis

• Community resource development:

  • Contribute to open repositories of validated protocols

  • Participate in collaborative projects establishing reference standards

  • Share detailed methodological notes beyond standard Materials and Methods sections

  • Develop and share positive and negative control materials

Researchers can significantly advance the field by adhering to these standardization practices and contributing to community resources that enhance reproducibility in PRKACA antibody applications across basic research and clinical contexts.