ACR6 Antibody

How to validate CCR6 antibody specificity in flow cytometry?

• Methodology:

  • Negative Controls: Use CCR6-knockout cell lines or tissues (e.g., CRISPR-edited models) to confirm absence of signal .

  • Isotype Controls: Compare staining with non-targeting IgG antibodies of the same host species .

  • Competition Assays: Pre-incubate antibodies with recombinant CCR6 protein to block binding .

• Key Data:

  Control Type	Purpose	Outcome Requirement
  Knockout cells	Specificity	No detectable signal
  Isotype control	Background noise	Signal ≤ 1% of test
  Competition assay	Epitope validation	≥80% signal reduction

What are optimal conditions for detecting CCR6 in Western blot?

• Protocol:

  • Sample Preparation: Use RIPA buffer with protease inhibitors to preserve CCR6’s extracellular domains .

  • Gel Electrophoresis: 10% SDS-PAGE for resolving CCR6 (~45 kDa) .

  • Blocking: 5% non-fat milk to reduce non-specific binding .

• Troubleshooting:

  • Weak bands may indicate epitope masking; try alternative lysis buffers (e.g., CHAPS detergent) .

How to select CCR6 antibodies for immunohistochemistry (IHC)?

• Criteria:

  • Epitope Accessibility: Prioritize antibodies targeting extracellular domains (e.g., N-terminal residues 1–50) .

  • Validation: Require peer-reviewed data showing CCR6 staining in Th17-cell-rich tissues (e.g., inflamed synovium) .

• Method:

  • Use antigen retrieval (citrate buffer, pH 6.0) for formalin-fixed samples .

How to resolve discrepancies in CCR6 expression data across studies?

• Analysis Framework:

  • Technical Variability: Compare antibody clones, fixation methods, and detection platforms (e.g., flow cytometry vs. IHC) .

  • Biological Context: Consider disease-specific CCR6 regulation (e.g., upregulated in rheumatoid arthritis vs. downregulated in HIV) .

  • Case Study: In psoriasis, CCR6+ Th17 cells infiltrate skin lesions but are rare in circulation .

What advanced methods enable epitope mapping of CCR6 antibodies?

• Techniques:

  • Alanine Scanning Mutagenesis: Identify critical residues for antibody binding .

  • Cryo-EM: Resolve antibody-antigen complexes to 3–4 Å resolution .

• Data Integration:

  Epitope Region	Functional Impact	Disease Relevance
  N-terminal loop	Blocks CCL20 binding	Autoimmunity
  Transmembrane domain	Alters signaling	Cancer metastasis

How to model CCR6/CCL20 axis perturbations in autoimmune disease?

• Experimental Design:

  • In Vivo Models: Use CCR6-KO mice crossed with autoimmune strains (e.g., EAE for multiple sclerosis) .

  • Antibody Blockade: Administer anti-CCR6 monoclonal antibodies (e.g., clone 53103.111) preclinically .

• Outcome Metrics:

  • Th17 cell migration (flow cytometry) .

  • IL-17/IL-22 levels (ELISA) .

Methodological Best Practices

• Antibody Validation Table :

  Application	Critical Controls	Acceptable Threshold
  Western Blot	Knockout lysate	No band in KO lane
  IHC	Isotype control	≤5% background staining
  Flow Cytometry	Fc receptor block	MFI ≤ 100 in controls

• Data Contradiction Workflow:

  • Replicate experiments across ≥3 biological replicates.

  • Cross-validate with orthogonal methods (e.g., RNAscope for CCR6 mRNA) .