Anti-ADAMTS-15 Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight band should I expect for ADAMTS-15 on Western blot?

ADAMTS-15 migrates at approximately 105-110 kDa on reducing SDS-PAGE, corresponding to the full-length 950-amino-acid protein. You may also observe a faster-migrating band around 85-90 kDa representing a processed form lacking the C-terminal ancillary domain. Because ADAMTS-15 is a secreted metalloprotease, band intensity will be strongest in conditioned media and extracellular matrix-enriched fractions. Detection in whole-cell lysates is typically weaker unless cells are actively secreting the enzyme during myogenesis or matrix remodeling.

What dilution should I start with for ADAMTS-15 Western blot?

We recommend starting at 1:1000 dilution for Western blot when using standard chemiluminescent detection. This dilution has been validated with human samples. Depending on ADAMTS-15 expression levels in your system, you may need to optimize between 1:500 and 1:2000. Secreted metalloproteases like ADAMTS-15 are often expressed at lower levels than cytosolic proteins, so concentrated conditioned media or matrix-enriched fractions typically yield stronger signals than whole-cell lysates. Include a titration series in your initial experiment to determine the optimal working concentration for your samples.

Will this antibody detect mouse or rat ADAMTS-15?

Cross-reactivity with mouse and rat ADAMTS-15 is predicted based on sequence homology but has not been experimentally validated. Human ADAMTS-15 shares approximately 85 percent sequence identity with mouse and rat orthologs in conserved domains. If you are working with rodent samples, we recommend testing the antibody at the same starting dilution (1:1000 for Western blot) and running a positive control with human recombinant ADAMTS-15 or lysate from a human cell line known to express the protease. Confirm specificity by siRNA knockdown or competition with immunizing peptide if available.

How should I prepare samples to detect ADAMTS-15 as a secreted protease?

Because ADAMTS-15 is secreted into the extracellular space, harvest conditioned serum-free media from cultured cells for 24-48 hours after reaching confluence. Concentrate media 10- to 20-fold using centrifugal filters (10 kDa cutoff) to enrich low-abundance secreted proteins. For tissue samples, consider extracting the extracellular matrix fraction with sequential salt and detergent buffers, as ADAMTS-15 associates with matrix components via its thrombospondin repeats. Include protease inhibitors that spare metalloproteases (avoid EDTA or chelators that strip zinc, which is required for structural integrity).

What are appropriate positive and negative controls for ADAMTS-15?

Use differentiating myoblast cultures (C2C12 or primary human myoblasts) as a positive control, since ADAMTS-15 is upregulated during myogenesis and facilitates cell fusion. Conditioned media collected during the differentiation phase typically shows robust expression. For tissue lysates, skeletal muscle undergoing regeneration or remodeling serves as a physiological positive control. Negative controls include non-muscle cell lines with minimal matrix remodeling activity or samples from cells transfected with ADAMTS-15-targeting siRNA. Recombinant human ADAMTS-15 protein provides an unambiguous molecular weight reference.

Does ADAMTS-15 have isoforms or splice variants I should know about?

The primary ADAMTS-15 transcript encodes the full-length 950-amino-acid protein. Minor splice variants have been reported in transcript databases, but functional characterization is limited and expression appears tissue-restricted. The most commonly observed variation on Western blot is proteolytic processing rather than alternative splicing. ADAMTS family members undergo post-translational cleavage that removes C-terminal ancillary domains, generating shorter active forms. If you observe multiple bands, consider that ADAMTS-15 may undergo autocatalytic or heterologous processing in the extracellular matrix, particularly in highly proteolytic environments.

Can I use this ADAMTS-15 antibody for immunohistochemistry or immunofluorescence?

This antibody has been validated for Western blot at 1:1000 dilution. Triple Point Biologics antibodies are routinely tested for IHC and immunofluorescence applications across the product line, but specific validation data for ADAMTS-15 in these formats should be confirmed with our technical support. For IHC, start at 1:100 to 1:200 dilution on formalin-fixed paraffin-embedded sections with heat-mediated antigen retrieval in citrate buffer. ADAMTS-15 localizes to the extracellular matrix and pericellular space, so expect diffuse extracellular staining in muscle and connective tissues rather than discrete intracellular compartments.

How should I store the ADAMTS-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 background. The 100 µg pack size is sufficient for approximately 100 Western blots at 1:1000 dilution. Once thawed, an aliquot remains stable at 4°C for up to one month if supplemented with 0.02 percent sodium azide or another antimicrobial preservative. For long-term storage beyond two years, keep at -80°C. Do not store in frost-free freezers where temperature cycling can denature the antibody over time.