PRKACA Antibody, HRP conjugated

What is PRKACA and why is it significant in research?

PRKACA (Protein Kinase A, alpha Catalytic subunit) is a critical enzyme involved in cAMP-dependent signaling pathways that regulates numerous cellular processes including metabolism, gene expression, and cell proliferation. Its significance in research spans multiple disease contexts:

• In fibrolamellar hepatocellular carcinoma (FLC), the DNAJB1-PRKACA fusion operates as a primary oncogenic driver

• Activating mutations in PRKACA have been identified in cortisol-producing adrenal tumors causing Cushing syndrome

• PRKACA overexpression mediates resistance to HER2-targeted therapy in breast cancer patients

The catalytic activity of PRKACA is essential for its biological functions, with phosphorylation at specific residues (particularly Thr197) regulating its enzymatic activity .

What are the key differences between various PRKACA antibodies available for research?

PRKACA antibodies vary based on several critical parameters that determine their research applications:

The selection of the appropriate antibody depends on the specific research question, with considerations for species cross-reactivity, particular domains of interest, and compatibility with experimental techniques.

How does an HRP-conjugated PRKACA antibody differ functionally from unconjugated versions?

HRP-conjugated PRKACA antibodies provide distinct advantages in certain research applications:

• Direct detection capability: The horseradish peroxidase enzyme directly conjugated to the antibody eliminates the need for secondary antibody incubation, reducing experimental time and potential background

• Simplified multiplexing: When combined with other fluorescent or enzymatic detection systems, HRP-conjugated antibodies enable cleaner multiplexing without cross-reactivity between secondary antibodies

• Enhanced sensitivity: Direct coupling of HRP can improve signal detection in applications like Western blotting and immunohistochemistry, particularly for low-abundance targets

• Specialized applications: Particularly valuable in techniques like ELISA and chromogenic immunohistochemistry where enzymatic amplification of signal is essential

The molecular weight of the conjugated antibody will be slightly higher than unconjugated versions, which should be considered when interpreting results.

What are the optimal protocols for using HRP-conjugated PRKACA antibodies in Western blotting?

The effective use of HRP-conjugated PRKACA antibodies in Western blotting requires careful optimization:

• Sample preparation considerations:

  • Include phosphatase inhibitors in lysis buffers to preserve phosphorylation states of PRKACA

  • Use appropriate detergents to solubilize membrane-associated PRKACA

  • Load sufficient protein (typically 20-40 μg for total cell lysates) to detect endogenous PRKACA

• Blocking and antibody incubation:

  • BSA-based blocking (3-5%) often performs better than milk for phospho-epitopes

  • Typical dilutions range from 1:500 to 1:2000 for HRP-conjugated antibodies

  • Incubate at 4°C overnight for maximum sensitivity or 1-2 hours at room temperature

• Detection optimization:

  • Select ECL substrate based on expected protein abundance

  • Perform brief (30-60 second) substrate incubation

  • Optimize exposure times to avoid signal saturation

When detecting the DNAJB1-PRKACA fusion protein, researchers have successfully used antibodies targeting the catalytic domain to distinguish between the 40 kDa wild-type PRKACA and the 46 kDa fusion protein .

How can researchers effectively employ PRKACA antibodies for immunohistochemistry and immunofluorescence studies?

Successful immunohistochemistry and immunofluorescence with PRKACA antibodies requires attention to several critical parameters:

• Tissue/sample preparation:

  • For FFPE tissues: Test multiple antigen retrieval methods (citrate buffer pH 6.0, EDTA pH 9.0)

  • For frozen sections: Brief fixation (10 min) in 4% paraformaldehyde often preserves epitopes

  • For cultured cells: Optimize fixation and permeabilization (0.1-0.3% Triton X-100) conditions

• Antibody optimization:

  • For HRP-conjugated antibodies: Block endogenous peroxidases with 0.3% H₂O₂ in methanol

  • Typical dilutions range from 1:100 to 1:500 for tissue sections

  • Include appropriate controls (isotype control, known positive tissues)

• Signal development:

  • For HRP-conjugated antibodies: Monitor DAB development microscopically to prevent overdevelopment

  • For dual staining: Apply HRP substrate before fluorescent detection

Researchers have successfully employed PRKACA immunohistochemistry to demonstrate increased expression in breast cancer samples following development of resistance to trastuzumab-containing therapy .

What are the most effective approaches for studying protein-protein interactions involving PRKACA?

Multiple techniques can be employed to investigate PRKACA interactions with regulatory subunits and other binding partners:

• Co-immunoprecipitation (Co-IP):

  • Use PRKACA antibodies to pull down protein complexes

  • Western blot for interacting partners like PRKAR1A

  • In wild-type conditions, PRKACA robustly pulls down PRKAR1A, while mutations like L206R disrupt this interaction

• Proximity-based methods:

  • Proximity ligation assay (PLA) enables visualization of protein interactions in situ

  • BioID or APEX2 proximity labeling can identify the broader PRKACA interactome

• Structural and functional validation:

  • Combine interaction studies with functional readouts of PKA activity

  • Correlate interaction disruption with biological consequences

Research has demonstrated that mutations in PRKACA can significantly alter its protein interactions, as exemplified by the L206R mutation which prevents binding to the regulatory subunit PRKAR1A, resulting in constitutive kinase activation in adrenal tumors .

How can PRKACA antibodies be used to investigate the oncogenic DNAJB1-PRKACA fusion in fibrolamellar carcinoma?

PRKACA antibodies provide critical tools for investigating this oncogenic fusion protein in multiple experimental contexts:

• Detection and validation approaches:

  • Antibodies targeting the PRKACA catalytic domain can detect both wild-type and fusion proteins

  • Junction-specific antibodies can provide selective detection of the fusion protein

  • Western blotting can distinguish the 46 kDa fusion protein from the 40 kDa wild-type protein

• Functional studies:

  • Monitor protein levels following fusion-specific knockdown approaches

  • In inducible shRNA systems, DNAJB1-PRKACA protein levels decrease within 24 hours of induction without affecting wild-type PRKACA expression

  • Assess the effects of targeted therapeutic approaches on fusion protein levels

• Translational applications:

  • Evaluate DNAJB1-PRKACA as a diagnostic biomarker

  • Investigate processing of fusion-derived peptides for immunotherapeutic approaches

  • Monitor response to targeted therapies in patient-derived models

Researchers have demonstrated that siRNAs conjugated to GalNAc can achieve specific knockdown of the DNAJB1-PRKACA fusion with high specificity (IC₅₀ of 1 pM), highlighting the therapeutic potential of targeting this oncogenic driver .

What role does PRKACA play in therapeutic resistance, and how can antibodies help elucidate these mechanisms?

PRKACA has emerged as a key mediator of resistance to targeted therapies, particularly in HER2-positive breast cancer:

• Expression and signaling analysis:

  • Immunohistochemistry reveals dramatically increased PRKACA expression in breast cancer samples following development of resistance to trastuzumab therapy

  • Western blotting demonstrates that PRKACA-mediated resistance operates independently of canonical HER2 signaling pathways (ERK/AKT)

• Mechanistic investigations:

  • PRKACA overexpression rescues HER2-amplified cells from lapatinib treatment through restoration of anti-apoptotic survival signaling

  • Unlike HRASV12-mediated resistance, PRKACA does not restore p-ERK or p-AKT levels, suggesting alternative survival mechanisms

• Clinical correlations:

  • High PRKACA expression is common in invasive breast cancers compared to normal breast tissue

  • Three of five matched patient samples showed increased PRKACA expression after developing resistance to anti-HER2 therapy

These findings suggest PRKACA as both a biomarker for resistance and a potential therapeutic target in combination strategies to overcome resistance to HER2-targeted therapies.

How can researchers use PRKACA antibodies to investigate post-translational modifications and their functional consequences?

PRKACA undergoes critical post-translational modifications that regulate its activity and localization:

• Phosphorylation analysis:

  • Phospho-specific antibodies targeting sites like Thr197 enable assessment of PRKACA activation status

  • Western blotting with phospho-specific antibodies before and after stimulation can reveal activation dynamics

  • Immunofluorescence with phospho-specific antibodies can reveal subcellular localization of active PRKACA

• Mass spectrometry approaches:

  • Immunoprecipitation with PRKACA antibodies followed by mass spectrometry can identify novel modification sites

  • Quantitative proteomics can reveal changes in modification patterns under different conditions

• Functional correlations:

  • Combine modification detection with kinase activity assays

  • Correlate modifications with protein-protein interactions and subcellular localization

Studies have demonstrated that the activating L206R mutation in PRKACA, found in cortisol-producing adrenal tumors, affects protein-protein interactions with regulatory subunits, providing insight into how structural changes impact PRKACA function .

What are common challenges when using PRKACA antibodies and how can researchers overcome them?

Researchers may encounter several challenges when working with PRKACA antibodies:

• Multiple bands in Western blotting:

  • Cause: May represent splice variants, post-translational modifications, or cross-reactivity

  • Solution: Validate with recombinant standards, genetic manipulation (siRNA knockdown), or phosphatase treatment for phosphorylated forms

• Weak or absent signals:

  • Cause: Low expression levels, epitope masking, or sample preparation issues

  • Solution: Optimize protein extraction methods, increase antibody concentration, extend incubation times, or use signal enhancement systems

• High background in imaging applications:

  • Cause: Insufficient blocking, high antibody concentration, or non-specific binding

  • Solution: Test alternative blocking agents (BSA vs. serum), optimize antibody dilution, increase wash steps

For specific applications like detecting the DNAJB1-PRKACA fusion protein, researchers have successfully optimized shRNA and siRNA approaches to achieve specific targeting of the fusion junction without affecting wild-type PRKACA expression .

How can researchers develop quantitative assays using PRKACA antibodies for biomarker applications?

Developing quantitative PRKACA assays requires careful methodological considerations:

• ELISA development:

  • Use capture/detection antibody pairs recognizing different epitopes

  • Include recombinant protein standards for absolute quantification

  • Validate assay parameters (sensitivity, specificity, reproducibility, dynamic range)

• Flow cytometry applications:

  • Optimize fixation and permeabilization for intracellular staining

  • Include isotype controls and fluorescence-minus-one (FMO) controls

  • Standardize with calibration beads for consistent quantification

• Digital pathology approaches:

  • Standardize staining protocols across multiple tissue samples

  • Develop algorithms for automated scoring of PRKACA expression

  • Correlate expression levels with clinical outcomes

Such quantitative approaches could be particularly valuable for monitoring PRKACA as a potential biomarker for therapeutic resistance in breast cancer, where increased expression has been associated with resistance to HER2-targeted therapies .

What considerations are important when designing immunotherapy approaches targeting the DNAJB1-PRKACA fusion protein?

The unique junction in the DNAJB1-PRKACA fusion creates neoantigens that can be targeted for immunotherapy:

• Neoantigen identification:

  • HLA-presented neoantigens specific to the DNAJB1-PRKACA fusion can be identified and characterized

  • Both HLA class I and class II ligands derived from the fusion can induce functional T cell responses

• T cell response characterization:

  • DNAJB1-PRKACA-derived peptides can induce multifunctional cytotoxic CD8+ and T-helper 1 CD4+ T cells

  • Single-cell RNA sequencing can identify multiple T cell receptors from fusion-specific T cells

• Therapeutic vaccination:

  • Vaccination with DNAJB1-PRKACA-derived peptides can induce persistent T cell responses

  • In a case report, vaccination induced multifunctional CD4+ T cells with an activated T-helper 1 phenotype and high T cell receptor clonality, accompanied by durable relapse-free survival for more than 21 months

These findings highlight the potential of targeting the DNAJB1-PRKACA fusion junction as a therapeutic strategy in fibrolamellar hepatocellular carcinoma.

How are PRKACA antibodies being used to develop novel therapeutic approaches?

PRKACA antibodies are facilitating multiple therapeutic development strategies:

• Targeted degradation approaches:

  • Antibodies help validate PRKACA degradation in response to proteolysis-targeting chimeras (PROTACs)

  • Detection of wild-type versus fusion proteins enables specificity assessment of degradation approaches

• RNA interference therapeutics:

  • GalNAc-conjugated siRNAs targeting the DNAJB1-PRKACA fusion junction show high potency (IC₅₀ of 1 pM) and specificity

  • Modifications such as phosphorothioate backbones and 5'-vinylphosphonate enhance circulation time and nuclease resistance

• Peptide vaccine development:

  • Vaccination with DNAJB1-PRKACA-derived peptides induces specific T cell responses

  • This approach has shown promise in a clinical case with durable relapse-free survival

The development of these targeted therapeutic strategies depends on high-quality antibodies for validation of target engagement and biological effects.

What are the latest findings regarding PRKACA's role in disease mechanisms beyond cancer?

While the search results focus primarily on oncogenic roles, PRKACA has broader implications in multiple disease contexts:

• Endocrine disorders:

  • Activating mutations in PRKACA cause cortisol-producing adrenal tumors and Cushing syndrome

  • The L206R mutation disrupts binding to regulatory subunits, leading to constitutive activation

• Therapeutic resistance mechanisms:

  • PRKACA overexpression mediates resistance to HER2-targeted therapies in breast cancer

  • Unlike other resistance mechanisms, PRKACA operates independently of canonical ERK/AKT pathways

• Novel signaling interactions:

  • Studies are revealing unexpected PRKACA interactions beyond the canonical PKA signaling pathway

  • These non-canonical interactions may explain disease-specific effects of PRKACA alterations

Understanding these diverse roles requires specific antibodies that can distinguish between different PRKACA forms and activation states in various cellular contexts.