AP-17 Antibody

Basic Research Questions

• What is ADAM17 and what are its key cellular functions?

  ADAM17 (A Disintegrin And Metalloprotease 17) is a multidomain transmembrane protein primarily known for releasing the active form of tumor necrosis factor (TNF)-α from its precursor, earning it the alternative name TACE (TNF-α Converting Enzyme) . The full-length protein contains 9 glycosylation sites, a signal peptide, propeptide, and exists in multiple isoforms produced by alternative splicing . While its calculated molecular weight is 93 kDa, observed molecular weight in some experimental contexts is 29 kDa, indicating potential post-translational processing .

  ADAM17 plays critical roles in:

  • Cytokine and growth factor shedding

  • Inflammatory signaling cascades

  • Development processes

  • Cancer progression through substrate cleavage

• What applications are validated for ADAM17-Specific antibody (20259-1-AP)?

  The ADAM17-Specific antibody (20259-1-AP) has been validated for multiple research applications:

  Application	Recommended Dilution	Positive Detection
  Western Blot (WB)	1:500-1:1000	Human placenta tissue
  Immunohistochemistry (IHC)	1:20-1:200	Human pancreas cancer tissue
  Immunofluorescence (IF)	See published data	Human samples
  ELISA	Application-specific	Human samples

  For IHC applications, antigen retrieval with TE buffer (pH 9.0) is recommended, though citrate buffer (pH 6.0) may serve as an alternative .

• How should researchers address the molecular weight discrepancy of ADAM17 in experimental settings?

  The significant difference between calculated (93 kDa) and observed (29 kDa) molecular weights of ADAM17 requires methodological considerations:

  • Confirm antibody specificity using appropriate positive controls (human placenta tissue is recommended)

  • Consider proteolytic processing and post-translational modifications affecting migration patterns

  • Use gradient gels to improve separation and resolution of protein isoforms

  • When analyzing western blot data, be aware that the 120-kDa form may represent the full-length protein while smaller bands may represent processed forms

  • Include denaturation controls to assess complex formation

Advanced Research Questions

• What is the relationship between IL-17 signaling pathways and ADAM17 in inflammatory conditions?

  IL-17 signaling and ADAM17 function intersect in several inflammatory contexts:

  • Both pathways are implicated in autoimmune conditions, with IL-17A/IL-17RA antibodies approved for treating psoriasis, psoriatic arthritis, and ankylosing spondylitis

  • IL-17 signaling is critical for mucosal immunity against opportunistic fungal pathogens like Candida albicans

  • ADAM17 processes membrane-bound TNF-α, which can modulate T cell responses including Th17 differentiation

  • In conditions like Antiphospholipid Syndrome (APS), imbalances between Th17 and T regulatory (Treg) cells contribute to pathogenesis, with increased Th17/Treg ratios

  Methodological approach: When investigating these pathways, researchers should consider measuring both ADAM17 expression/activity and IL-17 pathway components simultaneously to assess potential regulatory relationships.

• How can researchers effectively measure and interpret Th17/Treg imbalances in autoimmune conditions?

  Research on APS reveals important methodological considerations for studying T cell subset imbalances:

  • Use flow cytometry to detect absolute numbers of peripheral blood lymphocyte subsets and CD4+ T cell subsets

  • Employ flow cytometry bead arrays for measuring serum cytokine levels

  • Consider analyzing both cell populations and their associated cytokines (IL-17, IL-10)

  Key research findings demonstrate:

  • APS patients show significant decreases in NK cells (PAPS) and T, B, and CD4+T cells (SAPS)

  • Similar trends in CD4+T cell subsets occur in both PAPS and SAPS patients, including increased Th1 cells and decreased Th2 cells

  • Decreased Treg cells account for increased Th17/Treg ratios in APS patients compared to healthy controls

  • Cytokine levels correlate with clinical characteristics and autoantibody titers in APS patients

• What methodological approaches are recommended when studying IL-17 pathway effects on cancer immunotherapy response?

  Research on anti-PD-1 therapy reveals that activation of Th17 cells can undermine checkpoint inhibitor efficacy in certain cancer models:

  • Incorporate microbiota-sufficient and microbiota-deficient conditions, as lung macrophages from microbiota-sufficient mice can polarize naïve CD4+ T-cells to a Th17 phenotype

  • Measure downstream molecules like COX-2 and PGE2, which are enhanced by anti-PD-1 treatment

  • Include IL-17 neutralization conditions to assess pathway-specific effects (neutralization of IL-17 diminishes COX-2 and PGE2 induction)

  • Evaluate CTL (Cytotoxic T Lymphocyte) activity as a functional readout of immune response

  Experimental insight: Inhibition of COX-2 rescued CTL activity and restored tumor suppression in anti-PD-1-treated mice, revealing the molecular basis of IL-17-mediated resistance to checkpoint blockade .

• What are the optimal storage and handling conditions for ensuring ADAM17 antibody performance?

  For maximum stability and performance of ADAM17-Specific antibody (20259-1-AP):

  Storage Parameter	Recommended Condition
  Temperature	-20°C
  Buffer	PBS with 0.02% sodium azide and 50% glycerol pH 7.3
  Aliquoting	Unnecessary for -20°C storage
  Stability	One year after shipment when properly stored
  Special Note	20µl sizes contain 0.1% BSA

  Methodological recommendation: While aliquoting is not strictly necessary, it can prevent freeze-thaw cycles which may reduce antibody performance in sensitive applications like IHC at lower dilutions (1:20-1:50) .

• How should researchers approach experimental design when studying IL-17-dependent fungal immunity?

  When investigating IL-17's role in anti-fungal immunity:

  • Distinguish between effects of IL-17A and IL-17F blockade (IL-17A, but not IL-17F blockade, predisposes to oropharyngeal candidiasis)

  • Consider functional redundancy among IL-17 family cytokines

  • Recognize that IL-17R signaling is required for immunity to C. albicans in both mice and humans

  • Include appropriate models of opportunistic mucosal infections to assess IL-17-dependent defense mechanisms

  Clinical relevance: Patients receiving IL-17A or IL-17RA antibody therapies for autoimmune conditions may have increased susceptibility to opportunistic infections that require intact IL-17 signaling for host defense .

Methodology and Protocol Questions

• What optimization strategies should be employed when using MMP17 antibody (28429-1-AP) in different tissue types?

  Tissue-specific optimization for MMP17 antibody applications:

  Tissue Type	Application	Recommended Dilution	Notes
  Mouse brain	Western Blot	1:500-1:2000	Validated positive detection
  Rat brain	Western Blot	1:500-1:2000	Validated positive detection
  Human stomach cancer	IHC	1:50-1:500	Use TE buffer pH 9.0 for antigen retrieval

  Methodological recommendation: When working with new tissue types, begin with the middle of the recommended dilution range and adjust based on signal-to-noise ratio. Antibody titration is essential for optimal results, particularly in IHC applications .

• How can researchers effectively measure dynamic changes in IL-17/Treg balance in clinical samples?

  For longitudinal studies measuring IL-17/Treg dynamics:

  • Standardize collection timing relative to treatment interventions

  • Consider that peripheral blood samples may not provide sufficient material for in vitro experiments

  • Account for baseline medications that may affect immune cell populations

  • Use consistent markers for identifying Th17 (CD4+IL-17+) and Treg (CD4+CD25+FoxP3+) populations

  Research limitation awareness: Studies on APS patients show that different baseline medications and timing of thrombotic events may significantly impact peripheral blood lymphocyte subsets and cytokine levels .