Anti-ADAMTS-16 Rabbit Polyclonal Antibody

Frequently Asked Questions

What is the expected molecular weight for ADAMTS-16 on Western blot?

Full-length human ADAMTS-16 has a predicted molecular weight of approximately 133 kDa based on its 1224-amino acid sequence. However, ADAMTS proteases undergo proteolytic maturation involving propeptide cleavage, and ADAMTS-16 is a secreted glycoprotein, so you may observe the mature form running at a different apparent mass depending on post-translational modifications. In cell lysates, look for bands between 120-140 kDa. Conditioned media from expressing cells may show additional processed fragments if the protease undergoes autocatalytic cleavage. Always include a positive control lysate from ADAMTS-16-expressing cells to confirm expected migration.

What dilution should I start with for ADAMTS-16 Western blot?

We recommend starting at 1:1000 for Western blot with standard chemiluminescent detection. This dilution has been validated with the rabbit polyclonal format. If you are working with low-abundance samples such as serum or conditioned media, you may need to increase antibody concentration to 1:500. Conversely, if background is high with cell lysates overexpressing ADAMTS-16, titrate up to 1:2000. Blocking with 5% non-fat milk in TBST for one hour typically provides clean signal. Because ADAMTS-16 is a secreted protein, enriching conditioned medium by ultrafiltration often improves detection over whole-cell lysates.

Is this antibody validated for mouse ADAMTS-16 or only human?

This antibody has been validated for reactivity with human ADAMTS-16. Reactivity with mouse, non-human primate, and dog ADAMTS-16 is predicted based on epitope sequence conservation but has not been experimentally confirmed in our hands. Human and mouse ADAMTS-16 share approximately 80% amino acid identity, which suggests cross-reactivity is likely, but we recommend including appropriate positive and negative controls when working with non-human species. If you plan to use mouse tissue or lysates, consider testing the antibody on a recombinant mouse ADAMTS-16 control or known expressing cell line first.

Can I use this ADAMTS-16 antibody for immunohistochemistry?

Yes, Triple Point Biologics antibodies are validated for immunohistochemistry in addition to Western blot. ADAMTS-16 localizes to the extracellular matrix, so you should expect staining in stromal compartments, basement membranes, and pericellular regions depending on tissue type. We recommend antigen retrieval with citrate buffer, pH 6.0, for paraffin-embedded sections. Start with a dilution range of 1:100 to 1:200 for IHC. Because ADAMTS-16 is a secreted protein, signal intensity may vary depending on local matrix deposition and tissue fixation time. Include a negative control with rabbit IgG at equivalent concentration.

What positive control should I use for ADAMTS-16 Western blot?

ADAMTS-16 expression is relatively low in most tissues, which can complicate positive control selection. The protein is detected in placenta, kidney, and certain connective tissues, but commercial lysates may show weak signal. We suggest transiently transfecting HEK293T or COS-7 cells with a human ADAMTS-16 expression construct as a robust positive control. Harvest both cell lysate and conditioned medium 48 hours post-transfection; the secreted fraction usually yields stronger signal. If recombinant protein is available, 10-50 ng loaded per lane provides a useful reference. Avoid assuming high expression in untransfected cell lines without prior verification by RT-PCR.

Does ADAMTS-16 have multiple isoforms I should be aware of?

The UniProt entry for human ADAMTS-16 (Q8TE57) lists the canonical 1224-amino acid isoform as the primary product. Splice variants have been reported in transcript databases, but their expression at the protein level and functional significance remain poorly characterized in the literature. On Western blot, you are most likely to detect the full-length or mature processed form of the canonical isoform. If you observe multiple bands, consider that ADAMTS family members can undergo proteolytic maturation and domain shedding rather than representing true splice isoforms. Co-immunoprecipitation with domain-specific antibodies can help distinguish processing products from alternatively spliced forms.

How should I store the ADAMTS-16 antibody and what is the stability?

Store the antibody at –20°C in small aliquots to avoid repeated freeze-thaw cycles, which can reduce titer and increase aggregation. The 100 µg pack size is supplied in a stabilized formulation suitable for long-term storage at –20°C for at least 12 months from the date of receipt. For daily use, keep a working aliquot at 4°C for up to one month. Do not store diluted antibody in blocking buffer for more than one week, as proteases and microbial growth can degrade IgG. If you observe increased background or loss of signal over time, prepare a fresh aliquot from frozen stock.

Why am I not detecting ADAMTS-16 in my cell lysates?

ADAMTS-16 is a secreted extracellular matrix protein, so intracellular levels are often low unless you capture the biosynthetic pool before secretion. If you are preparing whole-cell lysates, most of the protein may have already been released into the culture medium. We recommend collecting conditioned serum-free medium after 24-48 hours of culture and concentrating it 10- to 20-fold using a 10 kDa cutoff centrifugal filter. Additionally, some cell types express very little endogenous ADAMTS-16. Confirm mRNA expression by qPCR before investing heavily in protein-level optimization. Including a protease inhibitor cocktail during lysis is essential to prevent degradation of ADAMTS family members.