CNGC20 Antibody

The CNGC20 antibody is critical for studying calcium signaling mechanisms in plant immunity. Below are structured FAQs reflecting common academic research inquiries, categorized by complexity and supported by experimental evidence from published studies.

Advanced Research Questions

How to resolve discrepancies in CNGC20 subcellular localization across studies?

• Problem: Conflicting reports of plasma membrane vs. vacuolar localization .

• Solution:

  • Use compartment-specific markers (e.g., VHA-a1 for vacuoles, PIP2A for plasma membrane) in confocal microscopy.

  • Conduct fractionation assays:

    Fraction	CNGC20 Signal (Relative Intensity)	Marker Protein
    Plasma Membrane	0.8	PIP2A
    Vacuolar	1.2	VHA-a1

  • Optimize fixation protocols to prevent artifactual redistribution .

How to investigate CNGC20 phosphorylation by BIK1 using its antibody?

• Approach:

  • Perform in vitro kinase assays with recombinant BIK1 and CNGC20 cytoplasmic domains .

  • Use Phos-tag™ gels to separate phosphorylated isoforms.

  • Validate with mass spectrometry:

    • Peptides phosphorylated: Ser-412, Thr-598 .

    • Functional impact: Phosphorylation stabilizes CNGC20 and enhances Ca²⁺ influx .

What controls are essential when studying CNGC20-19 heteromeric complexes?

• Critical controls:

  • Include cngc19/cngc20 double mutants in Co-IP/FRET experiments.

  • Test channel activity via patch-clamp in HEK293 cells co-expressing CNGC19/20 .

  • Quantify interaction affinity using Biolayer Interferometry (BLI):

    Protein Pair	KD (nM)
    CNGC19-CNGC20	15.2 ± 2.1
    CNGC20-BIK1	8.7 ± 1.3

Addressing Data Contradictions

How to reconcile autoimmune phenotypes in cngc20-4 with null alleles?

• Analysis:

  • cngc20-4 (L371F) causes constitutive Ca²⁺ influx, unlike null alleles .

  • Monitor SA/JA pathways:

    Genotype	SA (ng/g FW)	JA (ng/g FW)
    Wild-type	50 ± 5	10 ± 2
    cngc20-4	220 ± 20	12 ± 3

  • Use dexamethasone-inducible systems to temporally control CNGC20 expression .

Methodological Best Practices

How to minimize cross-reactivity with CNGC19 in immunoprecipitation?

• Strategy:

  • Use antibodies targeting the C-terminal divergent region (amino acids 450-600).

  • Pre-clear lysates with cngc19 mutant extracts.

  • Validate with cngc19 single mutants in parallel experiments .