Anti-ADAM-33 Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight should I expect for ADAM-33 on a Western blot?

Full-length ADAM-33 has a calculated molecular weight of approximately 90 kDa based on its 813-amino acid sequence. On reducing SDS-PAGE, you should expect a band near 100-110 kDa due to post-translational modifications, particularly N-glycosylation. ADAM-33 undergoes furin-mediated propeptide cleavage during maturation, so you may observe a mature form around 85-95 kDa depending on cell type and glycosylation status. If you detect lower molecular weight bands (60-70 kDa), these likely represent C-terminal fragments generated by autocatalytic processing or alternative translation initiation. Always include a positive control lysate from bronchial smooth muscle cells or vascular smooth muscle cells where ADAM-33 is abundantly expressed.

What dilution should I start with for Western blot detection of ADAM-33?

We recommend starting at 1:1000 dilution for Western blot applications based on validation data with this polyclonal antibody. This dilution typically provides a strong signal with low background when detecting endogenous ADAM-33 in human cell lysates. For samples with lower ADAM-33 expression, you may need to increase antibody concentration to 1:500, though this should be optimized against your specific lysate to avoid non-specific binding. Use 5% BSA or non-fat dry milk in TBST for blocking, and incubate overnight at 4°C. Because ADAM-33 is a membrane protein, ensure complete solubilization with RIPA or similar lysis buffer containing 1% NP-40 or Triton X-100.

Will this ADAM-33 antibody cross-react with mouse or rat samples?

Cross-reactivity with mouse and rat ADAM-33 is predicted based on sequence homology but not experimentally validated by us. Human ADAM-33 shares approximately 85% amino acid identity with mouse and 83% with rat orthologs, particularly in the metalloprotease and disintegrin domains where epitopes likely reside. If you are working with rodent samples, we recommend testing the antibody at the suggested 1:1000 dilution alongside a validated human positive control. Confirm any observed bands by including a blocking peptide control or siRNA knockdown. Monkey and dog cross-reactivity is also predicted due to high sequence conservation in primates and canines, but empirical validation in your system is essential.

What cell types or tissues show the highest ADAM-33 expression for positive controls?

ADAM-33 is most abundantly expressed in airway smooth muscle cells, bronchial smooth muscle, and vascular smooth muscle cells, making these ideal positive control sources. Human lung tissue lysates typically show robust ADAM-33 expression. In culture, primary human bronchial smooth muscle cells (HBSMC) or airway smooth muscle cell lines are reliable positive controls. ADAM-33 expression is notably lower or absent in epithelial cells and most hematopoietic lineages. For IHC validation, normal human lung sections provide excellent positive control tissue, with expected staining in smooth muscle layers surrounding bronchioles and blood vessels. Fibroblasts show variable but generally moderate expression depending on activation state.

Does ADAM-33 have splice variants I should be aware of when interpreting Western blots?

Yes, ADAM-33 has multiple splice variants arising from alternative splicing, with at least eight documented isoforms. The predominant full-length isoform is variant 1 (813 amino acids). Several shorter isoforms lack portions of the cytoplasmic tail or transmembrane domain due to exon skipping. Variant expression patterns differ between tissues and disease states, particularly in asthma-related remodeling. Because this antibody is raised against the propeptide domain (N-terminal region), it should recognize most variants that retain this epitope. However, variants with internal deletions may migrate differently on gels or show reduced immunoreactivity. If you observe multiple bands, confirm identity by comparing predicted sizes of known variants or performing peptide competition assays.

What sample preparation steps are critical for detecting membrane-bound ADAM-33?

Because ADAM-33 is a type I transmembrane metalloprotease anchored in the plasma membrane, complete protein extraction requires detergent-based lysis buffers. Use RIPA buffer or similar formulations containing 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS to solubilize membrane proteins effectively. Avoid mild lysis buffers designed for cytosolic extracts, as they will underestimate ADAM-33 levels. Include protease inhibitors (EDTA-free if preserving activity) and phosphatase inhibitors to prevent degradation. For cultured cells, scrape or trypsinize gently to preserve membrane integrity before lysis. Sonication or multiple freeze-thaw cycles can improve extraction efficiency. Load 20-40 µg total protein per lane for endogenous detection; overloading may increase background.

Can I use this antibody for immunofluorescence or IHC to visualize ADAM-33 localization?

Yes, Triple Point Biologics antibodies are validated for immunofluorescence and immunohistochemistry applications in addition to Western blot. For IF on cultured cells, use 1:100 to 1:200 dilution as a starting point. Because ADAM-33 localizes primarily to the plasma membrane, you should observe peripheral staining in smooth muscle cells or fibroblasts. Use methanol fixation or short paraformaldehyde fixation with appropriate permeabilization to preserve membrane antigens. For IHC on paraffin-embedded tissue sections, antigen retrieval with citrate buffer (pH 6.0) or Tris-EDTA (pH 9.0) is typically required. Expect positive staining in smooth muscle layers of airways and vessels. Optimize dilution and retrieval conditions empirically, as tissue processing affects epitope accessibility.

How should I store this ADAM-33 antibody and what is its shelf life?

Store the antibody at -20°C in single-use aliquots to avoid repeated freeze-thaw cycles, which can reduce titer and increase aggregation. Upon receipt, centrifuge briefly to collect contents, then aliquot into working volumes (10-20 µL recommended based on typical experiment needs at 1:1000 dilution). The antibody is supplied as a purified IgG fraction in phosphate-buffered saline, often with carrier protein and preservative. Under these conditions, stability is typically 12-24 months from manufacturing date when stored frozen. For short-term use (up to one month), diluted working stocks can be stored at 4°C with 0.02% sodium azide as preservative. Do not store diluted antibody in Tris buffers, as this may affect long-term stability.