PRKG2 Antibody

What is PRKG2 and what cellular functions does it regulate?

PRKG2 (also known as cGKII, PKG2) is a serine/threonine protein kinase activated by cGMP that plays crucial roles in several physiological pathways. It belongs to the cGMP-dependent protein kinase family and functions as a homodimer with a molecular weight of approximately 85-90 kDa . PRKG2 regulates:

• Intestinal secretion of water and electrolytes, particularly in response to E. coli toxin STa and the intestinal peptide guanylin

• Chondrocyte differentiation by coupling the cessation of proliferation and the start of hypertrophic differentiation through attenuating SOX9 function

• Alveolar type 2 epithelial (AT2) cell homeostasis and re-alveolarization of injured lungs

• Skeletal development and growth, with mutations associated with dwarfism in humans, cattle, and dog breeds

How should I select the appropriate PRKG2 antibody for my specific application?

Selection should be based on validated applications, species reactivity, and immunogen characteristics:

Always perform antibody titration in your specific experimental system to determine optimal conditions .

How can I optimize PRKG2 antibody performance in Western blotting?

For optimal western blot results with PRKG2 antibodies:

• Sample preparation: PRKG2 is predominantly expressed in brain tissue (mouse and rat) and specific cell types including intestinal and lung epithelial cells. Extract proteins using buffers containing phosphatase inhibitors to preserve phosphorylation status .

• Loading controls: Use appropriate loading controls based on tissue type (β-actin for general purposes, tissue-specific markers for specialized applications).

• Expected bands: Look for bands at 85-90 kDa for wild-type PRKG2. Note that some antibodies may detect bands at 100 kDa, which is different from the predicted 87 kDa molecular weight. This discrepancy may be due to post-translational modifications .

• Optimization protocol: Begin with a 1:1000 dilution in 5% BSA-TBST and optimize as needed. Incubate overnight at 4°C for best results .

What controls should I include when studying PRKG2 in functional assays?

For rigorous experimental design:

• Positive controls: Include mouse or rat brain tissue lysates for western blotting, which express high levels of PRKG2 .

• Negative controls: Consider using:

  • PRKG2 knockout or knockdown samples (gene editing or siRNA)

  • Isotype control antibodies for immunostaining applications

  • Secondary antibody-only controls to assess non-specific binding

• Inhibitor controls: Include PKG inhibitors like H89 (which also inhibits PKA) to confirm specificity of signaling pathway effects .

• Stimulation controls: Use cGMP analogs to activate PRKG2 and assess functional responses.

How can I effectively study PRKG2 in lung alveolar epithelial cells?

Recent research has demonstrated that PRKG2 regulates alveolar type 2 epithelial (AT2) cell homeostasis and differentiation . For studying PRKG2 in lung tissue:

• Cell isolation: Isolate primary AT2 cells from mouse lungs using dispase digestion (50 units/mL) followed by gentle mechanical disruption in DMEM/F-12 with 0.01% DNase .

• 3D organoid culture: Co-culture AT2 cells with lung fibroblasts in 3D Matrigel to study PRKG2's role in AT2 differentiation and organoid formation. Wild-type and PRKG2-knockout cells show distinct patterns of AT1 and AT2 marker expression .

• Immunofluorescence markers: Use anti-surfactant protein C (SFTPC) antibodies to identify AT2 cells and anti-podoplanin (PDPN) antibodies to identify AT1 cells in organoid structures .

• Functional readouts: Assess EdU incorporation to measure proliferation, which is altered in PRKG2-deficient cells compared to wild-type controls .

What are the best approaches for studying PRKG2 in skeletal development?

PRKG2 has been implicated in skeletal development, with mutations causing dwarfism in various species . For skeletal studies:

• Genetic analysis: Sequence the PRKG2 gene, particularly exon 15, which contains a nonsense mutation (R678X) in American Angus cattle with dwarfism that truncates 85 C-terminal amino acids of the kinase domain .

• Functional assays: Evaluate PRKG2's regulation of SOX9-mediated transcription of collagen genes (COL2 and COL10). Real-time PCR analysis shows increased expression of these genes when R678X mutant PRKG2 is expressed compared to wild-type PRKG2 .

• Cellular models: Use chondrocyte cell lines or primary growth plate chondrocytes to study PRKG2's role in growth plate development and endochondral ossification.

• In vivo models: Consider PRKG2 knockout mice or naturally occurring mutants to study skeletal phenotypes.

What are the common issues with PRKG2 antibodies and how can they be addressed?

How can I verify PRKG2 antibody specificity for my experimental system?

Verifying antibody specificity is crucial for reliable results:

• Genetic validation: Use PRKG2 knockout or knockdown samples as negative controls. Three published studies have used knockout/knockdown systems with PRKG2 antibodies to confirm specificity .

• Peptide competition: Pre-incubate the antibody with the immunizing peptide (if available) before application to demonstrate signal specificity.

• Multiple antibodies: Use antibodies raised against different epitopes of PRKG2 to confirm consistent results.

• Recombinant protein: Use purified recombinant PRKG2 protein as a positive control for western blotting.

• Mass spectrometry validation: Perform immunoprecipitation followed by mass spectrometry to confirm target identity.

How can PRKG2 antibodies be used to investigate cGMP/PKG signaling pathways in cancer?

Recent research has implicated PRKG2 in several cancer types:

• Cervical cancer: High-risk HPV16 E6 activates the cGMP/PKG pathway through glycosyltransferase ST6GAL1, where PRKG2 antibodies can help elucidate pathway components .

• Prostate cancer: EDNRB has been shown to negatively regulate glycolysis through the cGMP/PKG pathway, with implications for tumor suppression .

• Esophageal squamous cell carcinoma: Erianin exerts antineoplastic effects by activating the cGMP-PKG signaling pathway .

• Gastric carcinogenesis: Helicobacter pylori infection promotes carcinogenesis through ZEB1-upregulated PRTG via the cGMP/PKG signaling pathway .

Experimental approaches:

• Use PRKG2 antibodies in combination with phospho-specific antibodies to monitor pathway activation

• Perform co-immunoprecipitation to identify novel PRKG2 interacting partners in cancer cells

• Evaluate PRKG2 expression and localization changes during cancer progression using IHC/IF

What are the methodological considerations for studying PRKG2 protein-protein interactions?

PRKG2 functions through interactions with various partner proteins:

• Validated interaction partners: HSP90aA1 has been identified as an interactor of PRKG2 through binding ELISA assays .

• Co-immunoprecipitation protocol:

  • Use 0.5-4.0 μg of PRKG2 antibody for 1.0-3.0 mg of total protein lysate

  • Include appropriate detergents that preserve protein-protein interactions

  • Perform reciprocal co-IP with antibodies against suspected interaction partners

  • Validate interactions using proximity ligation assays or FRET approaches

• Activity-based interaction studies: Consider using active PRKG2 protein preparations for binding studies. In one reported approach, PRKG2 was diluted serially in PBS with 0.01% BSA, transferred to HSP90aA1-coated microtiter wells, and incubated for 2h at 37°C. After washing with PBST, anti-PRKG2 polyclonal antibody was applied followed by HRP-labeled secondary antibody and substrate solution .

How can PRKG2 antibodies be used to investigate genetic associations with skeletal phenotypes?

Recent genetic studies have identified PRKG2 polymorphisms associated with skeletal development:

• Association studies: PRKG2 gene polymorphisms have been linked to thoracolumbar vertebrae and carcass traits in Dezhou donkeys. Sanger sequencing can validate SNPs in the PRKG2 gene region, including g.162153251 G>A, g.162156524 C>T, and g.162158453 C>T .

• Antibody applications: PRKG2 antibodies can be used to:

  • Compare protein expression levels between different genotypes

  • Evaluate potential effects of SNPs on protein localization or stability

  • Assess downstream signaling pathway alterations in different genetic backgrounds

• Structure-function analysis: For mutations affecting the kinase domain (such as the R678X mutation in cattle), antibodies raised against different epitopes can help determine protein stability and localization patterns .

What considerations are important when optimizing a LanthaScreen kinase assay for PRKG2?

For researchers developing kinase activity assays for PRKG2:

• Optimization process requires three key steps:

  • Determining optimal kinase concentration at high ATP (1 mM)

  • Determining ATP Km,app (the EC50 value of ATP concentration)

  • Re-optimizing kinase concentration at ATP Km,app

• Assay detection: The LanthaScreen kinase assay uses TR-FRET emission ratios to measure activity, allowing for sensitive detection of inhibitor effects .

• Inhibitor testing: Once optimized, the assay can determine IC50 values for potential PRKG2 inhibitors, providing insights into structure-activity relationships.

• Advantages: This approach maximizes assay sensitivity toward both ATP-competitive and non-ATP-competitive inhibitors .