PRKX Antibody

What is PRKX and why would researchers need antibodies against it?

PRKX is a member of an ancient family of cAMP-dependent serine/threonine kinases that is phylogenetically distinct from other protein kinases. It plays critical roles in renal epithelial morphogenesis, macrophage and granulocyte maturation, and regulation of innate immune responses . PRKX antibodies are essential research tools for studying PRKX's expression patterns, subcellular localization, and functional roles in various biological processes. These antibodies enable researchers to track PRKX protein levels during developmental stages and in response to various stimuli .

What applications are PRKX antibodies typically validated for?

PRKX antibodies are validated for several research applications:

The choice of application depends on the specific research question. For example, IHC is typically used for studying tissue expression patterns, while WB is used for quantifying protein levels and molecular weight verification .

How can I verify the specificity of a PRKX antibody for my experimental model?

Antibody specificity validation is critical to ensure reliable results. Methods include:

• Molecular weight verification: Confirm that the antibody detects a protein band of approximately 41 kDa (the expected molecular weight of PRKX) in Western blotting .

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide to block specific binding.

• Knockout/knockdown controls: Use PRKX knockdown cells (as demonstrated with sh-bcPRKX-1 in studies ) or knockout models as negative controls.

• Cross-reactivity testing: Test the antibody on samples from different species if cross-reactivity is claimed (human, mouse, rat samples) .

• Immunogen sequence alignment: Compare the immunogen sequence with that of your species of interest to predict potential cross-reactivity .

What are the optimal conditions for PRKX immunohistochemistry?

For optimal PRKX detection in tissue sections, the following protocol has been validated:

• Tissue preparation: Fix tissues in an appropriate fixative (e.g., 4% paraformaldehyde).

• Antigen retrieval: Perform heat-induced epitope retrieval, typically with citrate buffer (pH 6.0) or EDTA buffer (pH 9.0).

• Blocking and permeabilization: Block with 10% normal goat serum in PBS, and permeabilize with 0.2-0.3% Triton X-100 .

• Primary antibody incubation: Incubate sections with anti-PRKX antibody (dilution 1:25-1:500 depending on the specific antibody) for 1 hour at room temperature or overnight at 4°C .

• Secondary antibody incubation: Use appropriate HRP-conjugated secondary antibodies, such as goat anti-rabbit F(ab')2 fragments .

• Visualization: Develop with DAB substrate kit, counterstain with hematoxylin, and dehydrate through an ethanol gradient and xylene before mounting .

How can I detect the subcellular localization of PRKX protein?

PRKX has been reported to localize in both cytoplasmic and nuclear compartments. To study its subcellular localization:

• Cell fractionation approach:

  • Separate cytosolic and nuclear fractions using standard protocols

  • Perform Western blotting on each fraction

  • Studies have detected PRKX in both cytosolic and nuclear extracts

• Immunofluorescence approach:

  • Transfect cells with PRKX expression constructs (or stain for endogenous PRKX)

  • Fix cells with 4% paraformaldehyde

  • Perform immunofluorescence staining

  • Analyze using confocal microscopy

• PRKX nuclear translocation studies:

  • Transfect cells with GFP-tagged PRKX (pEGFP/PRKX)

  • Treat with 100 μM 8-Br-cAMP at various time points (0, 1, 3, 5, 10, 15, 20, 30, 120 min)

  • Fix with 4% paraformaldehyde, wash with PBS/Tween, and examine by confocal microscopy

This approach allows for real-time visualization of cAMP-induced PRKX translocation events.

What are the recommended protocols for PRKX kinase activity assays?

To measure PRKX kinase activity:

• Immunoprecipitation-based kinase assay:

  • Transfect cells with FLAG-tagged PRKX

  • Lyse cells in appropriate buffer (e.g., 10 mM Tris-HCl, pH 7.2, 150 mM NaCl, 0.5% Triton X-100, 0.5% Tween-20 with protease inhibitors)

  • Immunoprecipitate with anti-FLAG antibody

  • Perform kinase assay using kemptide substrate (LRRASLG) in the presence of ATP (100 μM), magnesium (10 mM), and [γ-32P]ATP

  • Measure incorporation of 32P into the substrate

• Controls:

  • Include vector-transfected cells as negative controls

  • Use PKA inhibitors to confirm specificity

  • Consider using PKA-deficient cell lines (e.g., FIB4 cells) for cleaner results

How can I study PRKX's role in innate immune signaling?

PRKX has been implicated in regulating antiviral immune responses. To investigate this role:

• Knock-down/overexpression approaches:

  • Design shRNAs targeting PRKX (e.g., sh-PRKX-1 achieved 94% reduction in protein levels)

  • Transfect cells with PRKX expression plasmids for overexpression studies

  • Assess effects on viral replication and immune response genes

• Reporter assays:

  • Co-transfect cells with PRKX and luciferase reporters driven by IFN or NF-κB promoters

  • Measure luciferase activity to assess PRKX's impact on these signaling pathways

• Viral infection models:

  • Use relevant viral models (e.g., SVCV as demonstrated in research)

  • Measure viral titers using plaque assays

  • Assess expression of interferon-stimulated genes (ISGs) using qRT-PCR

• Stimulus-response experiments:

  • Treat cells with immunostimulants like LPS (1-50 μg/mL) or Poly(I:C) (5-50 μg/mL)

  • Infect cells with viruses at different MOIs (0.01-1)

  • Monitor PRKX expression changes over time (0-48h) using qRT-PCR

What is the relationship between PRKX and TAK1/IRF7 signaling?

Recent research has revealed PRKX's role in regulating the TAK1/IRF7 signaling pathway:

• Mechanism studies:

  • PRKX has been shown to down-regulate IFN promoter transcription but enhance NF-κB promoter transcription

  • PRKX suppresses TAK1/IRF7-mediated IFN induction

  • PRKX triggers lysosome-dependent degradation of TAK1

• Experimental approach:

  • Co-immunoprecipitation to detect PRKX-TAK1 interaction

  • Western blotting to monitor TAK1 protein levels in the presence/absence of PRKX

  • Use of lysosomal inhibitors to confirm the degradation mechanism

• Functional consequences:

  • PRKX knock-down enhances antiviral immunity by increasing ISG expression

  • PRKX overexpression increases susceptibility to viral infection

How does PRKX expression change during development and in response to stimuli?

PRKX expression is developmentally regulated and responsive to various stimuli:

• Developmental expression:

  • Use PRKX antibodies for immunohistochemical analysis of embryonic tissue sections

  • Perform Northern blot analysis of PRKX gene expression in fetal tissues

  • PRKX is expressed during metanephric kidney development but not in normal adult kidneys

• Stimulus-response experiments:

  • LPS treatment: PRKX expression increases immediately, then shows concentration-dependent patterns

  • Poly(I:C) treatment: PRKX expression increases after stimulation with pattern variations dependent on concentration

  • Viral infection response: Different viruses (e.g., SVCV vs. GCRV) trigger distinct temporal patterns of PRKX expression

• Experimental methodology:

  • Cell culture: Use appropriate cell lines (e.g., MPK cells for kidney research)

  • RNA isolation and qPCR analysis using PRKX-specific primers

  • Data analysis using the 2^-ΔΔCT method for relative quantification

Why might I observe non-specific bands when using PRKX antibodies in Western blotting?

Non-specific bands in Western blots can result from several factors:

• Antibody concentration: Excessive antibody can increase background and non-specific binding. Optimize by testing dilutions from 1:500 to 1:2000.

• Cross-reactivity: PRKX antibodies may cross-react with related proteins, especially other cAMP-dependent protein kinases. Check the antibody specificity data and consider using peptide blocking controls.

• Sample preparation: Insufficient denaturation or incomplete protein transfer can cause artifacts. Ensure complete sample denaturation and optimize transfer conditions.

• Blocking optimization: Insufficient blocking can cause background. Test different blocking reagents (BSA vs. non-fat dry milk) and durations.

• Secondary antibody optimization: Use highly cross-adsorbed secondary antibodies to reduce cross-reactivity. Consider testing different detection methods (chemiluminescence vs. fluorescence).

How can I optimize PRKX immunoprecipitation protocols?

For successful PRKX immunoprecipitation:

• Lysis buffer optimization:

  • Use a buffer containing 10 mM Tris-HCl (pH 7.2), 150 mM NaCl, 0.5% Triton X-100, 0.5% Tween-20, and protease inhibitors

  • Adjust detergent concentration based on PRKX's solubility in your specific cell type

• Antibody selection:

  • Use epitope-tagged PRKX (e.g., FLAG-tagged) for cleaner results

  • If using anti-PRKX antibodies, ensure they are validated for immunoprecipitation

  • Consider using antibodies conjugated to agarose or magnetic beads for efficient pull-down

• Protocol refinements:

  • Pre-clear lysates to reduce non-specific binding

  • Optimize antibody-to-lysate ratio (typically 1-5 μg antibody per mg of lysate)

  • Include multiple wash steps to reduce background

• Control experiments:

  • Include IgG control immunoprecipitations

  • Perform competition assays with immunizing peptide

  • Include input controls for quantitative comparison

What cell lines are recommended for studying endogenous PRKX expression?

Based on the research literature, several cell lines have been used successfully for PRKX studies:

• Kidney-derived cells:

  • HEK293 cells (human embryonic kidney cells)

  • MPK cells (Mylopharyngodon piceus kidney cells)

  • FIB4 cells (PKA-deficient renal epithelial cells) - useful for specific PRKX functional studies

• Other useful cell lines:

  • EPC cells (Epithelioma Papulosum Cyprinid cells)

  • CIK cells (Ctenopharyngodon idella kidney cells)

• Selection considerations:

  • Consider species compatibility with your antibody

  • Verify endogenous expression levels before experiments

  • For developmental studies, embryonic or fetal cell lines may be more appropriate

What are the emerging roles of PRKX in disease pathogenesis?

Recent research has highlighted PRKX's involvement in several disease processes:

• Kidney diseases:

  • PRKX is expressed during normal metanephric kidney development but not in the adult kidney

  • Aberrant PRKX expression has been observed in Autosomal Dominant Polycystic Kidney Disease (ADPKD)

  • Future research should investigate PRKX as a potential therapeutic target in renal pathologies

• Immune dysfunction:

  • PRKX regulates antiviral immune responses through the TAK1/IRF7 pathway

  • PRKX acts as a negative regulator of interferon production but enhances NF-κB signaling

  • This dual role suggests potential implications in autoimmune and inflammatory conditions

• Sex reversal disorders:

  • Abnormal recombination involving PRKX and related pseudogenes on chromosome Y can cause sex reversal disorders

  • This includes XX males and XY females

  • Further research is needed to understand the molecular mechanisms involved

How can I design experiments to study PRKX interactions with other signaling molecules?

To investigate PRKX's molecular interactions:

• Co-immunoprecipitation approaches:

  • Express tagged versions of PRKX and potential interacting partners

  • Perform reciprocal co-immunoprecipitations

  • Confirm results with endogenous proteins when possible

• Proximity ligation assay (PLA):

  • Useful for detecting protein-protein interactions in situ

  • Requires validated antibodies against PRKX and interacting partners

  • Provides spatial information about where interactions occur in cells

• Functional interaction studies:

  • Reporter assays to study effects on signaling pathways (e.g., IFN, NF-κB promoters)

  • Domain mapping through truncation mutants

  • Site-directed mutagenesis of key residues in PRKX

• Advanced techniques:

  • FRET/BRET analysis for real-time interaction studies

  • Mass spectrometry following immunoprecipitation for unbiased interaction discovery

  • Yeast two-hybrid screening for novel PRKX binding partners

What are the latest methodologies for studying PRKX phosphorylation targets?

To identify and validate PRKX phosphorylation targets:

• In vitro kinase assays:

  • Use recombinant PRKX and candidate substrates

  • Analyze with radioactive [γ-32P]ATP or phospho-specific antibodies

  • Kemptide (LRRASLG) has been validated as a PRKX substrate

• Phosphoproteomic approaches:

  • Compare phosphoproteomes in cells with PRKX overexpression vs. knockdown

  • Enrich phosphopeptides using TiO2 or IMAC techniques

  • Identify substrates using mass spectrometry

• Validation strategies:

  • Site-directed mutagenesis of candidate phosphorylation sites

  • Generation of phospho-specific antibodies

  • Functional assays to determine the biological significance of phosphorylation

• Computational prediction:

  • Use phosphorylation site prediction algorithms

  • Consider PRKX's preference for motifs similar to those of other cAMP-dependent kinases

  • Cross-reference with phosphoproteomic databases