Anti-ADAM-15 Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight should I expect for ADAM-15 on a Western blot?

Full-length ADAM-15 (Q13444) is an 863-amino acid protein with a predicted molecular weight of approximately 93 kDa. On reducing SDS-PAGE, you will typically observe bands between 100-110 kDa due to post-translational glycosylation of the extracellular domains. ADAM-15 undergoes proteolytic processing, so you may also detect lower molecular weight fragments corresponding to shed ectodomains or C-terminal fragments, particularly in cell culture supernatants or tissue samples with high protease activity. Always include a positive control lysate from cells known to express ADAM-15, such as HeLa or A549 cells.

What dilution should I start with for ADAM-15 Western blot and IHC?

For Western blot, begin at 1:1000 dilution in blocking buffer containing 5% non-fat milk or BSA in TBST. This dilution has been validated with this rabbit polyclonal antibody. For immunohistochemistry on paraffin-embedded sections, a typical starting range is 1:100 to 1:200 following heat-induced epitope retrieval in citrate buffer, pH 6.0. Optimal dilution will depend on your tissue type, fixation time, and detection system sensitivity. We recommend titrating across a dilution series with your specific sample type, as metalloproteinase expression varies widely across tissues and cell lines.

Is this ADAM-15 antibody specific to human or does it cross-react with mouse and rat?

This antibody has been validated for reactivity with human ADAM-15. Cross-reactivity with mouse and rat orthologs is predicted based on sequence homology but has not been experimentally validated. Human ADAM-15 shares approximately 85% identity with mouse and rat orthologs across the immunogenic region. If you are working with rodent samples, we recommend treating this antibody as experimental for those species and including appropriate positive controls from human cells alongside your rodent samples to assess specific versus cross-reactive signal. Request validation data for non-human species if available.

Does ADAM-15 require reducing conditions for Western blot detection?

ADAM-15 is a type I transmembrane protein with multiple disulfide bonds in its extracellular disintegrin and cysteine-rich domains. For optimal detection of the full-length protein by Western blot, perform SDS-PAGE under reducing conditions with DTT or beta-mercaptoethanol in your sample buffer. Under non-reducing conditions, intramolecular disulfide bonds may mask epitopes or alter migration. If you are specifically studying oligomerization or disulfide-linked ADAM-15 complexes, run parallel reduced and non-reduced gels. Standard RIPA or Laemmli buffer with reducing agent is suitable for most applications.

What tissues or cell lines are good positive controls for ADAM-15 expression?

ADAM-15 is broadly expressed but levels vary substantially. HeLa cervical carcinoma, A549 lung adenocarcinoma, and HEK293 cells show reliable endogenous expression and work well as positive controls for Western blot. For tissue samples, placenta, lung, and kidney express relatively high levels. ADAM-15 is upregulated in smooth muscle cells and endothelial cells during migration and wound healing, so these primary cultures can serve as functional positive controls. If your cell line shows no signal, consider stimulation with growth factors or use of an ADAM-15-transfected cell lysate as a reference standard.

Can this antibody detect the shed ectodomain of ADAM-15 in conditioned media?

ADAM-15 undergoes ectodomain shedding, releasing the extracellular portion into culture supernatants or extracellular fluids. Whether this antibody detects the shed ectodomain depends on the epitope location, which should be confirmed with the immunogen sequence. If the epitope resides within the metalloproteinase or disintegrin domains (extracellular), detection in concentrated conditioned media is possible. Concentrate serum-free conditioned media 10- to 20-fold using centrifugal filters before Western blot. If the epitope is within the cytoplasmic tail, only cell lysates will yield signal. Check immunogen details or contact us to confirm epitope mapping.

How should I store the ADAM-15 antibody and how long is it stable?

Store the antibody at -20°C in small aliquots to avoid repeated freeze-thaw cycles, which can reduce titer and increase aggregation. The antibody is supplied as a polyclonal rabbit serum or purified IgG in buffer containing carrier protein and preservative; check the product datasheet for exact formulation. Under these storage conditions, the antibody remains stable for at least 12 months from the date of receipt. For long-term storage beyond one year, -80°C is preferred. Once thawed, working aliquots can be kept at 4°C for up to one month if sodium azide is present. Do not store diluted antibody.

Why am I seeing multiple bands for ADAM-15 on my Western blot?

Multiple bands are common with ADAM-15 and reflect its biology. The protein exists as a full-length proform, a mature processed form after prodomain removal, and various shed or cleaved fragments. Heterogeneous glycosylation adds further complexity, producing a smeared or ladder-like appearance between 90-110 kDa for full-length forms. Lower molecular weight bands may represent the shed ectodomain, intracellular C-terminal fragments, or degradation products if protease inhibitors were insufficient during lysis. Always include a protease inhibitor cocktail in your lysis buffer. If bands persist, validate identity by including a blocking peptide control or testing lysate from ADAM-15 knockout cells.