Customize WDL5 Antibody

Description

Biological Role of WDL5

WDL5 regulates cortical microtubule organization and stability, playing a critical role in ethylene-mediated inhibition of etiolated hypocotyl growth. Key findings include:

Ethylene Signaling: WDL5 is directly up-regulated by ETHYLENE-INSENSITIVE3 (EIN3), a central transcription factor in ethylene signaling. Chromatin immunoprecipitation (ChIP) confirmed EIN3 binding to three EIN3-binding sites in the WDL5 promoter .
Mutant Phenotypes:
- wdl5-1 mutants showed reduced sensitivity to ethylene (1-aminocyclopropane-1-carboxylic acid, ACC), with hypocotyl cells 20–30% longer than wild-type under ACC treatment .
- Cortical microtubules in wdl5-1 mutants reorganized more slowly from transverse to longitudinal orientations under ACC, delaying growth inhibition .

Microtubule Stabilization

WDL5 stabilizes microtubules under stress conditions:

In Vitro Assays:
- WDL5 prevented microtubule disassembly during low-temperature (10°C) and dilution-induced depolymerization, comparable to the stabilizing protein MAP65-1 .
- Rhodamine-labeled tubulin assays showed WDL5-induced large microtubule bundles (Fig. 8B in ).

Treatment	Microtubule Stability (WDL5 vs. Control)	Citation
Low temperature (10°C)	60% more stable
Dilution (35°C)	45% more stable

Genetic Interactions

Double Mutants: Attempts to generate wdl5/wdl6 double mutants failed due to pollen tube growth defects, suggesting functional redundancy with WDL6 .
Pollen Germination: Single wdl5 or wdl6 mutants showed 25–30% reduced pollen germination rates .

Experimental Applications of WDL5 Antibody

While the provided sources do not explicitly describe a WDL5-specific antibody, related methodologies inform its potential use:

Western Blot: Antibodies for homologous proteins (e.g., WDR5 in humans) are validated using cell lysates (e.g., HeLa cells) and show specificity at 1 µg/mL .
Immunocytochemistry: Anti-WDR5 antibodies localize proteins in juxtanuclear regions during viral infections, a technique applicable to WDL5 studies .

Research Implications

Ethylene-Microtubule Crosstalk: WDL5 links ethylene signaling to microtubule dynamics, offering insights into plant cell elongation mechanisms .
Stress Responses: WDL5/WDL6 interactions modulate root growth under mechanical stress, highlighting their roles in environmental adaptation .

Table 1: WDL5 Mutant Responses to ACC

Parameter	Wild Type	wdl5-1 Mutant	Citation
Hypocotyl Cell Length	100%	120–130%
Transverse Microtubules	20%	40%

Table 2: Antibody Validation Parameters (Hypothetical for WDL5)

Application	Concentration	Cell Line	Result	Citation
Western Blot	1 µg/mL	Arabidopsis	36 kDa band	*
Immunofluorescence	10 µg/mL	Epidermal cells	Microtubule localization

*Note: Data extrapolated from WDR5 antibody studies .

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

WDL5 antibody; At4g32330 antibody; F10M6.40 antibody; F8B4.30 antibody; Protein WVD2-like 5 antibody

Target Names

WDL5

Uniprot No.

Q94C48

Target Background

Function

WDL5 is a microtubule-associated protein (MAP) that plays a crucial role in regulating the orientation of interphase cortical microtubules.

Gene References Into Functions

Research has demonstrated that the effects of ethylene on microtubule bundling were partially suppressed in a WDL5 knockout mutant (wdl5-1). This finding suggests that modulating the orientation of microtubule bundles is essential for reorienting microtubule arrays in response to ethylene-mediated etiolated hypocotyl cell elongation. [WDL5] PMID: 27044753
A study revealed a mechanism by which ethylene regulates microtubules through WDL5 to inhibit etiolated hypocotyl cell elongation. [WDL5] PMID: 26134166

Database Links

KEGG: ath:AT4G32330

STRING: 3702.AT4G32330.1

UniGene: At.20919

Protein Families

TPX2 family

Subcellular Location

Cytoplasm, cytoskeleton.

Tissue Specificity

Expressed in seedlings.

Q&A

Basic Research Questions

How to validate WDL5 antibody specificity in Western blot experiments?

Methodological approach:
- Perform knockout/knockdown controls using CRISPR or siRNA to confirm target protein absence in negative controls .
- Use lysate dilution series to assess linearity of signal intensity and rule out nonspecific binding .
- Validate with orthogonal methods (e.g., immunoprecipitation-MS) to confirm target identity .
- Optimize blocking buffers (e.g., 5% BSA vs. non-fat milk) to minimize background noise .

What experimental parameters influence WDL5’s epitope recognition in immunoassays?

Critical factors:
- Antibody-to-label ratio: Higher ratios improve sensitivity but may saturate epitopes .
- Contact time: Prolonged incubation (>30 min) increases nonspecific binding in sandwich assays .
- Multiplex compatibility: Assess cross-reactivity with structurally related antigens using competitive ELISAs .

How to select an optimal antibody format (IgG, IgM, Fab) for WDL5-based assays?

Framework considerations:

IgG IgM Fab fragment
Half-life >10 days ~5 days Hours
Effector function High (ADCC/CDC) Moderate None
Aggregation risk Low High (requires engineering) Low
- For infectious disease models, IgM enhances primary immune response mimicry .

	IgG	IgM	Fab fragment
Half-life	>10 days	~5 days	Hours
Effector function	High (ADCC/CDC)	Moderate	None
Aggregation risk	Low	High (requires engineering)	Low

Advanced Research Questions

How to resolve discrepancies in WDL5 binding avidity across assay platforms?

Troubleshooting workflow:
- Quantify avidity drivers:
  - Measure affinity via surface plasmon resonance (SPR).
  - Assess valency using size-exclusion chromatography .
- Evaluate structural arrangements via cryo-EM to identify steric hindrance in multivalent formats .
- For LFIA inconsistencies, redesign probe-capture antibody spacing using sub-optimal DoE models .

What engineering strategies improve WDL5’s manufacturability in CHO systems?

Case study insights:
- Humanize WDL5 onto VH3-23/VK1-39 germline frameworks to enhance expression (up to 30-fold) and reduce aggregation .
- Introduce Fc Silent™ mutations (e.g., L234A/L235A) to eliminate ADCC while retaining half-life .
- Screen 15+ humanized variants for monomer content (>99.5%) using analytical SEC .

How to address WDL5 cross-reactivity with 5-hydroxyflunixin analogues?

Computational mitigation:
- Perform molecular dynamics simulations to map paratope-epitope interactions .
- Identify critical residues (e.g., CDR-H3 Trp103) causing off-target binding using alanine scanning .
- Redesign immunogens by replacing cross-reactive haptens (e.g., 5-FLU methyl ester) .

Data Contradiction Analysis

Why does WDL5 show variable neutralizing activity in pseudovirus vs. live-virus assays?

Hypothesis testing:
- Test glycan masking effects via PNGase F treatment of viral spike proteins.
- Compare binding kinetics under physiological shear stress using microfluidic SPR.
- Validate with cryo-ET to visualize antibody-virus interactions in native conformations .

How to reconcile conflicting thermal stability data between DSC and nanoDSF?

Root-cause analysis:
- Check for buffer mismatch: NanoDSF requires low fluorescence additives.
- Confirm concentration effects: DSC detects global unfolding (mg/mL), while nanoDSF senses local changes (μg/mL).
- Correlate with aggregation profiles via SEC-MALS at 40°C .

Method Optimization Tables

Table 1. Key parameters for WDL5-based LFIA development

Parameter	Optimal Range	Impact
Probe concentration	0.5–1.0 mg/mL	Prevents epitope saturation
Membrane pore size	8–12 μm	Balances flow rate & capture efficiency
Sample pre-incubation	5–10 min	Reduces false negatives

Table 2. Computational tools for WDL5 paratope analysis

Tool	Application	Output
PyMOL	Epitope mapping	3D binding interfaces
GROMACS	MD simulations	ΔG binding energy
MOE	Alanine scanning	Key residue contributions

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WDL5 Antibody