Anti-ADAM-17 Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight band should I expect for ADAM-17 on Western blot?

Full-length ADAM-17 migrates at approximately 100-120 kDa on Western blot, reflecting the mature glycosylated form of the 824-amino acid protein. The predicted molecular weight from sequence is around 93 kDa, but post-translational modifications including N-glycosylation shift the apparent size upward. You may observe additional bands corresponding to pro-ADAM-17 (approximately 110-120 kDa) or processed fragments depending on cell type and activation state. Treatment with PNGase F can help confirm glycosylation contributions. The cytoplasmic tail can also be subject to proteolytic processing, occasionally producing lower molecular weight species in some cell lines.

What starting dilution should I use for Western blot with this ADAM-17 antibody?

We recommend starting at 1:1000 dilution for Western blot applications with standard chemiluminescent detection. This dilution has been validated in our lab using human cell lysates. Optimal dilution may vary depending on ADAM-17 expression levels in your specific cell type or tissue, so you may need to titrate between 1:500 and 1:2000. Samples with high endogenous ADAM-17 expression, such as activated macrophages or certain tumor cell lines, often work well at 1:1500 or 1:2000. Always include a positive control lysate from a known ADAM-17-expressing cell line during initial optimization.

Will this antibody recognize mouse and rat ADAM-17?

This antibody is validated for human ADAM-17 and shows predicted cross-reactivity with mouse, rat, and pig based on epitope sequence alignment. Human and mouse ADAM-17 share approximately 88% sequence identity overall, with higher conservation in the catalytic and disintegrin domains. We recommend empirical validation on your specific mouse or rat samples, starting with the 1:1000 dilution used for human lysates. If signal is weak, you may need to adjust to 1:500. A positive control from mouse macrophages (RAW 264.7) or rat smooth muscle cells can help confirm cross-reactivity in your hands.

Does ADAM-17 expression change with cell stimulation, and how does that affect antibody detection?

ADAM-17 is constitutively expressed in most cell types but its activity and sometimes total protein levels increase following stimulation with phorbol esters, LPS, TNF-alpha, or other inflammatory signals. The antibody recognizes ADAM-17 regardless of activation state, so you should detect both inactive and active forms. However, stimulated cells may show increased signal intensity and potentially altered banding patterns due to increased maturation of the pro-form or enhanced trafficking to the plasma membrane. If comparing basal versus stimulated conditions, PMA treatment (100-200 ng/ml, 30-60 minutes) serves as a useful positive control for ADAM-17 upregulation in many cell types.

What lysis buffer and sample preparation work best for detecting ADAM-17?

ADAM-17 is a transmembrane protein, so efficient extraction requires detergent-based lysis buffers. RIPA buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) works reliably for total cell lysates. Include protease inhibitors (EDTA-free if you plan downstream metalloprotease assays) and phosphatase inhibitors if examining regulatory phosphorylation. For membrane-enriched preparations, 1% Triton X-100 in TBS is sufficient. Avoid boiling samples in reducing sample buffer for extended periods; 5 minutes at 95°C is adequate. ADAM-17 is stable during standard sample handling, but avoid repeated freeze-thaw cycles of lysates.

What positive and negative control cell lines should I use for ADAM-17 Western blots?

HeLa, HEK293, and THP-1 cells express readily detectable levels of endogenous ADAM-17 and serve as reliable positive controls. THP-1 monocytes differentiated with PMA show particularly strong ADAM-17 signal. For a negative control, consider using lysate from ADAM-17 knockout cells if available, or perform a blocking experiment with the immunizing peptide if provided. Alternatively, pre-treat a duplicate blot with ADAM-17 siRNA-transfected lysate as a knockdown control. TNF-alpha-stimulated cells often show enhanced ADAM-17 levels and can help confirm antibody specificity when compared to unstimulated controls from the same cell type.

Can this antibody detect the soluble shed ectodomain of ADAM-17?

This polyclonal antibody is raised against the extracellular region and should theoretically recognize shed ADAM-17 ectodomain fragments. However, soluble ADAM-17 ectodomain is typically present at much lower concentrations in conditioned media or serum compared to membrane-associated full-length protein in lysates. Detection may require concentrating conditioned media (via centrifugal filters or immunoprecipitation) and loading larger volumes. The shed ectodomain typically migrates around 80-90 kDa. For reliable detection of soluble ADAM-17, we recommend starting with cell lysates to confirm antibody performance, then optimizing concentration and loading conditions for conditioned media applications.

How should I store this ADAM-17 antibody and what is its shelf life?

Store the antibody at -20°C in its original formulation. Avoid repeated freeze-thaw cycles, which can reduce titer and increase aggregation over time. For frequent use, aliquot the stock into single-use volumes (10-20 µL) immediately upon receipt. Aliquots stored at -20°C typically retain activity for at least two years from manufacture date. For short-term use within four weeks, diluted working stocks can be stored at 4°C with 0.02% sodium azide as preservative, though we generally recommend preparing fresh dilutions in blocking buffer for each experiment. Do not store diluted antibody in azide-free buffer at 4°C for more than one week.