ADAMTS18 Antibody

What is ADAMTS18 and what are its key functional domains?

ADAMTS18 is a secreted Zn-metalloproteinase that belongs to the ADAMTS family. Its structure includes a signal peptide, pro-domain, metalloprotease domain, disintegrin domain, central TS-1 domain, Cys-rich domain, spacer domain, and TS-1 like repeat domain . This protease enzyme plays crucial roles in extracellular matrix remodeling and has been implicated in various biological processes including inflammation, tissue repair, and tumor progression . ADAMTS18 has been involved in development, hemostasis, and various malignancies, suggesting its multifunctional nature in physiological and pathological contexts .

What molecular weights should researchers expect when detecting ADAMTS18?

When working with ADAMTS18 antibodies in Western blot applications, researchers should expect to detect multiple molecular weight bands. Studies using tissues from Adamts18 knockout mice have identified five significantly attenuated bands located at 180, 135, 95, 72, and 45 kDa . These bands represent different forms of ADAMTS18 that potentially exist in tissues, likely resulting from post-translational modifications, proteolytic processing, and splice variants. The calculated molecular weight of ADAMTS18 is approximately 135 kDa , but observed molecular weights can vary depending on the specific antibody used and the tissue or cell type being examined.

What applications are ADAMTS18 antibodies typically used for?

ADAMTS18 antibodies have been validated for multiple research applications including:

• Western blot (WB) with recommended dilutions of 1:500-1:2000

• Immunohistochemistry (IHC) with recommended dilutions of 1:50-1:300

• Enzyme-linked immunosorbent assay (ELISA) with dilutions of 1:2000-1:5000

• Immunocytochemistry (ICC)

These antibodies enable the detection and analysis of ADAMTS18 in various cell types and tissue samples, providing valuable insights into its functions and mechanisms of action .

What tissue and cell types are known to express ADAMTS18?

ADAMTS18 expression has been documented in several tissues:

• Kidney: Expression is observed in developing kidneys, with levels gradually increasing as the kidney continues to mature during embryonic development . It plays a role in ureteric epithelium and branch tip development .

• Lung: ADAMTS18 is expressed in developing lungs and influences the timing of specialized cell type differentiation .

• Brain: Immunohistochemistry studies have detected ADAMTS18 in human brain tissue .

• Cancer tissues: ADAMTS18 has been studied in various cancer contexts, including human cervical cancer and has been identified as a potential tumor suppressor .

How can researchers validate the specificity of ADAMTS18 antibodies?

Validating antibody specificity is crucial for reliable research outcomes. For ADAMTS18 antibodies, researchers should employ multiple approaches:

• Genetic models: Using tissues from Adamts18 knockout (KO) mice as negative controls is the gold standard approach . This allows identification of non-specific binding.

• Multiple applications testing: Validate the antibody across different applications (WB, IHC, ELISA) to ensure consistent results .

• Peptide blocking: Pre-incubating the antibody with its immunizing peptide should abolish specific staining .

• Multiple antibody comparison: Using different antibodies targeting distinct epitopes of ADAMTS18 can confirm specificity. For example, studies have developed six murine monoclonal antibodies against different functional regions of ADAMTS18 .

• Expected molecular weight confirmation: Verifying that detected bands match the expected molecular weights for ADAMTS18 (180, 135, 95, 72, and 45 kDa) .

What methodological considerations are important when studying ADAMTS18's role in kidney development?

ADAMTS18 plays a significant role in kidney development, particularly in ureteric bud (UB) branching and immune microenvironment regulation. Key methodological approaches include:

• Gene expression analysis: RT-PCR can be used to track ADAMTS18 expression levels at different developmental stages .

• Immunofluorescence staining: Using UB markers such as E-cadherin alongside ADAMTS18 antibodies helps visualize branching defects .

• In vitro organ culture: Microisolation of embryonic kidney tissues (E14.5d) cultured in DMEM allows for experimental manipulation .

• Gene knockdown approaches: Microinjection of ADAMTS18 KO cells into the posterior renal mesenchyme can reveal developmental impacts .

• Flow cytometry: This technique enables analysis of immune cell infiltration (B cells, CD8+ T cells, neutrophils) in embryonic kidney tissues following ADAMTS18 knockdown .

• Molecular pathway analysis: Western blotting for analyzing expression of immune checkpoints (PD-1/PD-L1 and CTLA-4) that may be activated following ADAMTS18 knockdown .

Research has shown that ADAMTS18 gene deletion significantly reduces UB branching and increases the proportion of B cells and CD8+ cells in embryonic kidneys, suggesting that ADAMTS18 regulates kidney development through immune microenvironment modulation .

How can different ADAMTS18 antibodies help distinguish between protein variants?

Different forms of ADAMTS18 (resulting from post-translational modifications, cleaved forms, and splice variants) can be distinguished using targeted antibody approaches:

• Domain-specific antibodies: Using antibodies targeting different functional domains allows identification of specific protein regions present in different forms. Research has developed monoclonal antibodies against various functional regions of ADAMTS18 .

• N-terminal vs. C-terminal antibodies: Two specific monoclonal antibodies (N-3 and C-5) have been shown to precisely identify different molecular weight bands of ADAMTS18 .

• Comparison with knockout controls: Using tissues from Adamts18 knockout mice helps identify which bands are specific to ADAMTS18 .

• Inhibition assays: Studies using antibodies against the C-terminal portion of ADAMTS18 have provided evidence that this region might contain key functional properties .

These approaches collectively enable researchers to resolve the post-translational modification status, cleaved forms, and splice variants of ADAMTS18 that previously proved difficult to characterize .

What are the optimal storage and handling conditions for ADAMTS18 antibodies?

Proper storage and handling of ADAMTS18 antibodies are crucial for maintaining their functionality:

• Temperature considerations:

  • Long-term storage: -20°C for up to one year

  • Short-term storage: 4°C for up to one month

  • Avoid repeated freeze-thaw cycles

• Buffer components:

  • Typical storage buffers contain pH 7.4 PBS, 40-50% glycerol, 0.02-0.05% sodium azide, and sometimes 0.5% BSA

  • For specialized applications requiring conjugation, BSA-free and sodium azide-free formulations may be necessary

• Alternative formulations:

  • For conjugation chemistry, special formulas with trehalose and/or glycerol can provide protection without interfering with conjugation

  • Small aliquots should be prepared to minimize freeze-thaw cycles

• Carrier considerations:

  • For conjugation to biotin or other molecules, carrier-free antibody preparations are preferred

  • When storing carrier-free antibodies at -20°C, cryoprotectants like glycerol are recommended

How does ADAMTS18 contribute to lung development, and what methodologies best study this function?

ADAMTS18 plays a role in lung development, particularly influencing the timing of specialized cell type differentiation. Key methodological approaches include:

• Expression analysis: Whole-mount analysis and immunostaining can be used to examine ADAMTS18 expression patterns during lung development .

• Cell marker studies: Immunostaining with markers such as Sox2 (proximal epithelial progenitor), Sox9 (distal epithelial progenitor), Muc5ac (mucosal cell), Sftpc (alveolar epithelial type 2), and Tuba4a (ciliated cell) can reveal how ADAMTS18 affects cell differentiation .

• Quantitative PCR: This technique allows precise measurement of gene expression changes in wildtype versus ADAMTS18 mutant lungs .

• Cell proliferation and apoptosis analysis: EdU incorporation and Caspase 3 immunostaining can determine if ADAMTS18 affects cell proliferation or death in developing lungs .

Research has shown that loss of Adamts18 in embryonic lungs significantly delays the appearance of specialized cell types in both proximal and distal positions of the developing airways, without affecting early proximal-distal regionalization or cell proliferation/apoptosis .

What cross-reactivity concerns exist when using ADAMTS18 antibodies?

When working with ADAMTS18 antibodies, researchers should be aware of potential cross-reactivity issues:

• Family member homology: ADAMTS18 shares high sequence similarity with ADAMTS16, another family member . This homology can lead to cross-recognition between ADAMTS18 and homology domains of other family members .

• Species considerations: While many ADAMTS18 antibodies react with both human and mouse ADAMTS18 , cross-reactivity with other species (e.g., pig) requires experimental validation .

• Validation approaches:

  • Testing the antibody in tissues from ADAMTS18 knockout mice is the most definitive approach to confirm specificity

  • Comparing expression patterns using multiple antibodies targeting different epitopes

  • Pre-absorption with recombinant proteins of other ADAMTS family members

• Application-specific considerations: Cross-reactivity may differ between applications (WB vs. IHC), requiring optimization for each technique .

How can ADAMTS18 antibodies be modified for specialized research applications?

For specialized research applications, ADAMTS18 antibodies can be modified in several ways:

• Biotin conjugation: Useful for amplification techniques and streptavidin-based detection systems. This requires carrier-free antibody preparations without BSA or sodium azide .

• Buffer exchange: For applications sensitive to specific buffer components, antibodies can be reformulated. For example, removal of sodium azide may be necessary for certain enzymatic assays or live cell applications .

• Storage modification: For frequent use, small aliquots stored at 4°C with appropriate preservatives can minimize freeze-thaw cycles .

• Specialized formulations: Custom formulations with trehalose or alternative stabilizers can support specific applications like conjugation chemistry while maintaining antibody stability .

• Fragmentation: For certain applications, Fab or F(ab')2 fragments may provide better tissue penetration or reduced background.

When planning antibody modifications, researchers should discuss specific requirements with antibody manufacturers, as many offer custom formulation services for specialized applications .

Common ADAMTS18 Antibody Specifications

Molecular Weight Forms of ADAMTS18 Detected in Various Tissues

ADAMTS18 Expression in Development and Disease