Anti-ADAMTS-9 Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight should I expect for ADAMTS-9 on a Western blot?

Full-length ADAMTS-9 runs at approximately 220 kDa, corresponding to the 1935-residue precursor. You may observe additional bands near 200 kDa representing processed forms after pro-domain cleavage. Because ADAMTS-9 is a secreted extracellular matrix protein, conditioned media typically yields stronger signal than whole-cell lysates. Glycosylation heterogeneity can produce diffuse banding rather than sharp single bands. If you detect lower molecular weight species below 150 kDa, consider whether proteolytic processing or degradation has occurred during sample preparation. Running a parallel sample with protease inhibitors helps distinguish biological processing from artifact.

What dilution should I start with for Western blot detection of ADAMTS-9?

We recommend starting at 1:1000 dilution for Western blot applications. This concentration has been validated with human samples and provides a reasonable balance between signal and background. If your ADAMTS-9 expression level is low or you are working with conditioned media diluted during collection, begin with a more concentrated antibody at 1:500 and titrate upward if background is acceptable. Conversely, if you are enriching extracellular matrix proteins or using overexpression systems, you may achieve adequate signal at 1:2000. Overnight incubation at 4°C often improves sensitivity compared to shorter room-temperature protocols.

Is this antibody validated for mouse and rat samples?

The antibody has been sequence-validated against human ADAMTS-9 and should recognize mouse and rat orthologs based on epitope conservation, but we classify these as predicted rather than empirically validated reactivities. Mouse ADAMTS-9 shares approximately 88% identity with human, and the conservation is high in structured domains. If you are working with rodent samples, plan to run a positive control such as tissue known to express ADAMTS-9 (growth plate cartilage, aorta) alongside your experimental samples. Titre the antibody as you would with a new target, starting at the recommended 1:1000 dilution for Western blot.

Can I use this antibody for immunohistochemistry on cartilage or vascular tissue?

Yes, the antibody is validated for IHC and ADAMTS-9 is abundant in cartilage extracellular matrix and vascular smooth muscle, making these appropriate tissues. Antigen retrieval is typically required because ADAMTS-9 is embedded in proteoglycan-rich matrix. Citrate buffer at pH 6.0 with heat-induced epitope retrieval works well for most formalin-fixed paraffin-embedded tissues. Start with a 1:100 to 1:200 dilution for IHC and optimize based on signal-to-noise in your specific tissue. Hyaluronidase or chondroitinase pre-treatment may improve accessibility in heavily glycosylated cartilage sections. Include a no-primary-antibody control to assess non-specific binding in matrix-rich regions.

What is the best positive control tissue or cell line for ADAMTS-9 expression?

Chondrocytes and growth plate cartilage express ADAMTS-9 constitutively and serve as reliable positive controls. Human chondrosarcoma cell lines such as SW1353 express detectable levels. For vascular research, aortic smooth muscle and atherosclerotic plaques show expression. If working with conditioned media, confirm secretion by comparing lysate and media fractions from the same culture; ADAMTS-9 should be enriched in media. Versican-producing fibroblasts often co-express ADAMTS-9 for localized proteoglycan remodeling. Gene expression databases confirm high expression in mesenchymal tissues, so primary MSCs or differentiated chondrocytes are practical bench-level positive controls.

Does ADAMTS-9 have isoforms or splice variants I should know about?

The major characterized isoform is the full-length 1935-residue form. UniProt documents shorter variants arising from alternative splicing, but functional data on these remain limited. Most published studies focus on the canonical secreted metalloproteinase. The enzyme undergoes furin-mediated pro-domain cleavage during secretion, so you may detect both pro-form and mature processed species depending on your sample source. If you observe multiple bands on Western blot, consider that these likely represent post-translational processing rather than distinct splice isoforms. For critical experiments distinguishing isoforms, pair antibody detection with RT-PCR using isoform-specific primers.

Should I collect conditioned media or cell lysates for ADAMTS-9 detection?

Conditioned media typically provides stronger signal because ADAMTS-9 is a secreted extracellular matrix metalloproteinase. Collect serum-free conditioned media after 24 to 48 hours of culture, then concentrate 10- to 20-fold using centrifugal filters with a 100 kDa cutoff to enrich large glycoproteins. Include protease inhibitors immediately upon collection to prevent degradation. Cell lysates contain intracellular ADAMTS-9 in the secretory pathway but at lower abundance. If comparing secretion efficiency or testing trafficking mutants, run paired lysate and media samples on the same gel. For extracellular matrix studies, decellularized matrix preparations capture the biologically active pool.

How should I store the antibody and what is the expected shelf life?

Store the antibody at –20°C as supplied. For routine use, prepare small aliquots to avoid repeated freeze-thaw cycles, which can reduce titer over time. Once thawed, an aliquot remains stable at 4°C for approximately two weeks; add 0.02% sodium azide as preservative if you plan to keep a working stock at 4°C longer. Do not store diluted antibody in blocking buffer for more than a few days. Rabbit polyclonal antibodies maintained under these conditions typically retain activity for two years from manufacturing date. If you notice increased background or decreased signal over time, compare results with a freshly thawed aliquot before troubleshooting other variables.