Customize PLDALPHA4 Antibody

Description

Role in Stress Signaling

PLDALPHA4 is implicated in plant stress responses, particularly under salicylic acid (SA) induction. Studies using this antibody revealed:

SA-induced PLD Activation: PLDALPHA4 activity increases 2.5-fold within 60 minutes of SA treatment, producing phosphatidylbutanol (PBut) enriched in 16:0/18:2 and 16:0/18:3 acyl chains .
Membrane Heterogeneity: PLDALPHA4-generated PA species localize to membrane subdomains, suggesting compartmentalized signaling roles .

Enzymatic Specificity

Substrate Selectivity: In vitro assays showed PLDALPHA4 activity on phosphatidylcholine (PC) and phosphatidylethanolamine (PE) substrates, with no preference for acyl chain composition .
Calcium Dependence: Unlike PLDζ isoforms, PLDALPHA4 requires calcium for activity, aligning with its role in stress-responsive pathways .

Western Blot Validation

Expected Molecular Weight: 91.8 kDa (precursor form) .
Specificity: No cross-reactivity with PLDBETA1 or other Arabidopsis PLD isoforms confirmed via knockout mutant analysis .

ELISA and Immunofluorescence

Optimal Dilution: 1:1000 for WB; titrations required for IF/IHC .
Limitations: Serum-based formulation may require blocking with non-specific IgGs to reduce background .

Comparative Insights

PLDALPHA4 vs. Other PLD Isoforms

Feature	PLDALPHA4	PLDBETA1	PLDEPSILON
Gene Locus	AT1G55180	AT2G42010	AT1G55180 (alternative splice variant)
Calcium Requirement	Yes	No	Yes
Stress Response	SA, oxidative stress	Abiotic stress	Hormone signaling
Subcellular Local.	Plasma membrane	Cytoplasmic vesicles	Nuclear envelope

Future Directions

CRISPR/Cas9 Knockouts: PLDALPHA4 antibody aids in validating gene-edited Arabidopsis lines lacking functional PLDALPHA4 .
Transgenic Studies: Tagged PLDALPHA4 variants (e.g., GFP fusions) enable real-time tracking of enzyme localization .

Product Specs

Buffer

**Preservative:** 0.03% Proclin 300
**Constituents:** 50% Glycerol, 0.01M PBS, pH 7.4

Form

Liquid

Lead Time

Made-to-order (14-16 weeks)

Synonyms

PLDALPHA4 antibody; PLDEPSILON antibody; At1g55180 antibody; F7A10.25 antibody; Phospholipase D alpha 4 antibody; AtPLDalpha4 antibody; PLD alpha 4 antibody; EC 3.1.4.4 antibody; PLDalpha3 antibody; Phospholipase D epsilon antibody; AtPLDepsilon antibody; PLD epsilon antibody

Target Names

PLDALPHA4

Uniprot No.

Q9C888

Target Background

Function

PLDALPHA4 is a phospholipase D that hydrolyzes glycerol-phospholipids at the terminal phosphodiesteric bond, producing phosphatidic acids (PA). This enzyme promotes growth and plays a role in nitrogen signaling.

Gene References Into Functions

Research indicates that increased expression of PLD epsilon has the potential to enhance crop plant growth and yield under both nitrogen-deficient and nitrogen-sufficient conditions. PMID: 26260942
Overexpression of PLD-epsilon leads to increased plant growth, root development, and biomass accumulation in response to nitrogen availability. PMID: 19143999

Database Links

KEGG: ath:AT1G55180

STRING: 3702.AT1G55180.1

Protein Families

Phospholipase D family, C2-PLD subfamily

Subcellular Location

Cell membrane.

Tissue Specificity

Expressed in roots, leaves, stems, siliques,flowers and inflorescences.

Q&A

Basic Research Questions

How to validate PLDALPHA4 antibody specificity in novel experimental systems?

Method: Combine immunogen affinity purification (as described for polyclonal antibodies ) with orthogonal validation techniques:
- Perform western blotting against recombinant PLDALPHA4 and deletion mutants to confirm target band specificity.
- Use siRNA knockdown or CRISPR-Cas9 knockout models to verify signal reduction in cellular assays .
- Cross-reference with BLAST homology analysis to rule out off-target binding to homologous proteins .

What protocols optimize PLDALPHA4 antibody performance in multiplex immunoassays?

Recommendations:
- Pre-adsorb antibodies against common blocking agents (e.g., recombinant BSA ).
- Validate cross-reactivity using antigen-coated microarrays with related phospholipase D family members.
- Optimize dilution factors through checkerboard titrations (start with 1:100–1:500 range ).

How to resolve contradictory immunohistochemistry results across tissue types?

Troubleshooting workflow:

Step	Action	Rationale
1	Compare fixation methods (PFA vs. methanol)	Epitope accessibility varies by tissue preparation
2	Validate with RNAscope® or qPCR	Confirm protein-RNA expression correlation
3	Perform competitive inhibition with recombinant PLDALPHA4	Specificity confirmation

Advanced Research Questions

What computational strategies predict PLDALPHA4 antibody-antigen binding dynamics?

Structural modeling:
- Use ABodyBuilder2 for 3D paratope prediction .
- Perform molecular dynamics simulations focusing on CDR loop flexibility (critical for phospholipase D interactions ).
- Validate with SPR measurements of kon/koff rates (target Kd < 10 nM for high-affinity binding ).

How to design longitudinal studies assessing PLDALPHA4 antibody clinical utility?

What statistical approaches resolve multi-antibody dataset contradictions?

Advanced analysis:

Method	Application	PLDALPHA4 Case Example
Super Learner	Integrate Random Forest/SVM classifiers	Predict clinical outcomes from 20+ biomarkers
PCA dimensionality reduction	Identify dominant variance components	Separate technical vs. biological variability
Finite mixture models	Account for heterogeneous subpopulations	Resolve bimodal antibody response distributions

Data Conflict Resolution Table

Conflict Type	Resolution Strategy	Supporting Evidence
Variable inter-lab quantification	Standardize against WHO reference sera	Malaria antibody harmonization protocols
Epitope mapping discrepancies	Combine hydrogen-deuterium exchange MS with cryo-EM	PfEMP1 antibody validation precedent
Batch-to-batch variability	Implement ISO 13485-compliant QC with ≥3 biological replicates	HIT antibody manufacturing standards

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PLDALPHA4 Antibody