Anti-ADAMTS-10 Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight should I expect for ADAMTS-10 on Western blot?

Full-length ADAMTS-10 has a predicted molecular weight of approximately 120 kDa based on the 1103-amino acid sequence. However, because ADAMTS-10 is a secreted glycoprotein with multiple potential N-glycosylation sites, the apparent molecular weight on Western blot typically ranges from 130 to 150 kDa depending on post-translational modifications. You may also observe lower molecular weight bands corresponding to processed fragments, as ADAMTS family proteases can undergo autocatalytic cleavage or other proteolytic processing in the extracellular space. Conditioned media from expressing cells often shows more pronounced ladder patterns than whole-cell lysates.

What starting dilution should I use for ADAMTS-10 Western blot?

We recommend starting at 1:1000 for Western blot applications based on validation with human samples. This dilution has produced clear signal with acceptable background in our hands. Because ADAMTS-10 is a secreted protein, signal intensity will depend heavily on expression level and whether you are analyzing cell lysates versus conditioned media. For conditioned media, you may achieve better results at 1:2000 if expression is robust. If working with tissue lysates where ADAMTS-10 abundance is lower, begin at 1:500 and titre upward. Always include a known positive control such as fibroblast lysate or appropriate tissue extract.

Is this antibody validated for mouse and rat ADAMTS-10?

This antibody has been validated for human ADAMTS-10. Cross-reactivity with mouse and rat is predicted based on sequence homology but has not been experimentally confirmed in our laboratory. Human ADAMTS-10 shares approximately 85 percent identity with mouse and rat orthologs, with conservation highest in the metalloprotease and disintegrin-like domains. If you plan to use mouse or rat samples, we recommend confirming specificity with a positive control lysate from cells known to express ADAMTS-10, and consider including a blocking peptide or knockout control if available. Non-human primate reactivity is also predicted given high sequence conservation.

Can I use this antibody for immunofluorescence on fixed cells?

Yes, Triple Point Biologics antibodies are validated for immunofluorescence applications. Because ADAMTS-10 is secreted into the extracellular space, expect staining in the extracellular matrix surrounding expressing cells rather than intracellular signal, unless you are examining biosynthetic compartments in high-expressing cells. Methanol fixation often preserves extracellular matrix epitopes better than paraformaldehyde for ADAMTS family proteins. Start with a 1:100 to 1:200 dilution and include appropriate controls: cells with known low or absent ADAMTS-10 expression, and secondary antibody-only controls to assess non-specific matrix binding, which can be significant in ECM-rich samples.

What positive control samples work best for ADAMTS-10 Western blot?

Primary human dermal fibroblasts and skin tissue lysates serve as reliable positive controls, as ADAMTS-10 is expressed in connective tissues involved in microfibril assembly. Ocular tissues, particularly ciliary body and trabecular meshwork, also express ADAMTS-10 at detectable levels. Among cell lines, check the literature for models relevant to your research question; expression is often context-dependent and not uniformly high across standard immortalized lines. When working with conditioned media, concentrate samples 10- to 20-fold using centrifugal filters with appropriate molecular weight cutoffs to enhance detection. Include molecular weight markers spanning 100 to 250 kDa to capture full-length and processed forms.

Does ADAMTS-10 have splice variants I should know about?

The canonical ADAMTS-10 transcript encodes the 1103-amino acid isoform and is the predominant form reported in the literature. Minor splice variants have been deposited in sequence databases, but their functional significance and tissue distribution remain poorly characterized. Most studies reference the full-length form that includes the signal peptide, prodomain, metalloprotease, disintegrin-like, thrombospondin type-1 repeats, cysteine-rich, and spacer domains. If you observe multiple bands on Western blot, proteolytic processing is a more likely explanation than alternative splicing. Consider testing samples under reducing versus non-reducing conditions, as disulfide-linked complexes with fibrillin or other binding partners may alter migration.

How should I prepare samples to detect secreted ADAMTS-10?

ADAMTS-10 is secreted into the extracellular space and associates with microfibrils, so conditioned media and extracellular matrix preparations yield stronger signal than whole-cell lysates. Collect serum-free conditioned medium after 24 to 48 hours of culture, then concentrate 10- to 20-fold using centrifugal concentrators with a 30 kDa molecular weight cutoff. Avoid repeated freeze-thaw cycles, as ADAMTS proteases can be sensitive to degradation. For tissue samples, include protease inhibitors during extraction and consider protocols that solubilize extracellular matrix proteins. Comparing secreted versus cell-associated fractions can clarify retention versus efficient secretion in your system.

What are the recommended storage conditions for this antibody?

Store the antibody at –20°C in small aliquots to avoid repeated freeze-thaw cycles, which can reduce titre and increase aggregation. The product is supplied as a polyclonal antiserum in a buffered solution. Once thawed, an aliquot can be held at 4°C for up to one month for routine use. For long-term storage beyond six months, –80°C is preferable. Addition of glycerol to a final concentration of 50 percent prevents freezing at –20°C and allows liquid storage, which is convenient for frequently used antibodies. Sodium azide at 0.02 to 0.05 percent can be added as a preservative for 4°C storage but may interfere with HRP-based detection systems.