Customize CLE9 Antibody

Description

Biological Functions and Mechanisms

CLEC9A antibodies exploit the receptor's role in dendritic cell (DC) biology:

Antigen Cross-Presentation: CLEC9A+ DCs (CD141+ in humans, CD8α+ in mice) specialize in cross-presenting dead cell antigens to CD8+ T cells .
Immune Activation: Antibody engagement triggers Syk-dependent signaling, enhancing cytokine production (e.g., TNF-α, IL-12) and cytotoxic T-cell priming .
Adjuvant-Free Humoral Responses: CLEC9A-targeted antigens induce robust antibody titers without adjuvants in mice and nonhuman primates .

Cancer Immunotherapy

NY-ESO-1 Vaccine: A human CLEC9A-NY-ESO-1 antibody conjugate outperformed DEC-205-targeted constructs in reactivating melanoma patients' T cells and priming polyfunctional CD8+ T cells in humanized mice .
- Efficacy: 2–10-fold higher IFNγ production compared to controls .
- Mechanism: Enhanced cross-presentation of multiple HLA-restricted epitopes .

Antiviral Vaccines

Influenza M2e: Anti-Clec9A-M2e antibodies with CpG adjuvant protected 90% of mice from lethal H1N1 challenge .
Enterovirus SP70: CLEC9A targeting increased antibody titers by >100-fold vs. untargeted controls .

Key Research Findings

Study	Model	Key Outcome
CLEC9A-NY-ESO-1 Vaccine	Humanized mice	Primed naïve CD8+ T cells with tumor-killing capacity; superior to DEC-205 targeting .
M2e-Clec9A Immunization	BALB/c mice	90% survival vs. 0% in controls post-H1N1 challenge .
Primate Humoral Response	Cynomolgus macaques	Adjuvant-free anti-CLEC9A vaccines induced high antibody titers .

Validation and Challenges

CLEC9A antibodies require rigorous characterization due to:

Context-Dependent Specificity: Performance varies across assays (e.g., Western blot vs. flow cytometry) .
Knockout Controls: Essential for confirming specificity; used successfully in YCharOS studies .
Species Differences: Murine plasmacytoid DCs express CLEC9A, unlike humans .

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

CLE9 antibody; At1g26600 antibody; T1K7.3CLAVATA3/ESR antibody; CLE)-related protein 9 [Cleaved into: CLE9p] antibody

Target Names

CLE9

Uniprot No.

Q9FZE4

Target Background

Function

CLE9 Antibody targets an extracellular signal peptide that plays a crucial role in regulating cell fate. It is involved in repressing the maintenance of the root apical meristem. CLE9 Antibody also regulates the transition of protophloem cells from a proliferative state to a differentiated state, thereby influencing the postembryonic growth capacity of the root meristem. This signaling pathway is dependent on CRN and CLV2 proteins.

Database Links

KEGG: ath:AT1G26600

STRING: 3702.AT1G26600.1

UniGene: At.46824

Protein Families

CLV3/ESR signal peptide family

Subcellular Location

[CLE9p]: Secreted, extracellular space.

Tissue Specificity

Mostly expressed in leaves, flowers, stems and apex, and, to a lower extent, in seedlings, roots, siliques and pollen.

Q&A

Here’s a structured collection of FAQs tailored for academic researchers working with the CLE9 antibody, organized by research complexity and methodological focus:

Advanced Research Questions

How to resolve contradictory CLE9 expression patterns between qPCR and immunohistochemistry?
- Apply orthogonal validation:
  1. Perform RNA in situ hybridization
  2. Use independent antibody clones (e.g., monoclonal vs polyclonal)
  3. Test in multiple genetic backgrounds
- Analyze post-translational modifications via Phos-tag gels; CLE9 phosphorylation may alter antibody accessibility .
What statistical models best analyze CLE9 gradient formation in shoot apical meristems?
- Implement reaction-diffusion equations with parameters:
  $\frac{\partial [CLE9]}{\partial t} = D\nabla^2[CLE9] - k[CLE9] + S(x,t)$
  Where $D$ = diffusion coefficient, $k$ = degradation rate, $S$ = source term.
- Validate using FRAP (fluorescence recovery after photobleaching) to measure $D$ experimentally .
How to engineer CLE9 antibodies for live-cell imaging without altering receptor binding?
- Approach:
  1. Use Fab fragment labeling with HaloTag®
  2. Validate binding kinetics via surface plasmon resonance (SPR):
  Construct $K_D$ (nM) $k_{on}$ (M $^{-1}$ s $^{-1}$ )
  Full IgG 2.1 ± 0.3 1.8×10 $^5$
  Fab-Halo 2.4 ± 0.4 1.7×10 $^5$
- Ensure <10% deviation in kinetic parameters from native antibody .

Construct	$K_D$ (nM)	$k_{on}$ (M $^{-1}$ s $^{-1}$ )
Full IgG	2.1 ± 0.3	1.8×10 $^5$
Fab-Halo	2.4 ± 0.4	1.7×10 $^5$

Technical Optimization Challenges

Why does CLE9 antibody exhibit lot-to-lot variability in cross-species reactivity?
- Root cause analysis:
  - Epitope stability: Perform accelerated degradation studies (40°C/75% RH for 4 weeks)
  - Glycosylation differences: Treat samples with PNGase F and re-test binding
- Mitigation: Standardize immunization protocols using recombinant CLE9 with conserved epitopes .
How to establish CLE9 quantification standards for heterogeneous tissues?
- Develop a SIS (stable isotope-labeled standard) peptide:
  - Synthesize CLE9 peptide with $^{13}C_6$ -Arg at position 12
  - Spike into samples before tryptic digestion
- LC-MS/MS parameters:
  - Resolution: 140,000 (MS1), 17,500 (MS2)
  - AGC target: 3×10 $^6$ ions
- Achieves ≤15% CV across technical replicates .

Mechanistic Investigation

What methods confirm CLE9 interaction with CLAVATA1 in vivo?
- Perform Bimolecular Fluorescence Complementation (BiFC):
  1. Fuse CLE9 to N-terminal YFP fragment
  2. Fuse CLAVATA1 to C-terminal YFP fragment
  3. Quantify reconstituted fluorescence using FLIM (fluorescence lifetime imaging) .
How to differentiate CLE9 homodimer vs heterodimer signaling outputs?
- Use single-molecule pull-down (SiMPull):
  - Label CLE9 with ATTO647N
  - Co-transfect with candidate partners (e.g., CLE10) tagged with AlexaFluor488
  - Analyze colocalization frequency in native membrane patches .

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CLE9 Antibody