Anti-ADAMTS-6 Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight should I expect for ADAMTS-6 on a Western blot?

Full-length ADAMTS-6 has a predicted molecular weight of approximately 123 kDa based on the 1,117 amino acid sequence (UniProt Q9UKP5). However, because ADAMTS-6 is a secreted glycoprotein with extensive post-translational modifications, the apparent molecular weight often runs higher, typically between 130-150 kDa on reducing SDS-PAGE. You may also observe lower molecular weight bands representing proteolytic processing fragments, which is common for ADAMTS family members. The pattern can vary depending on cell type and whether you are analyzing lysate, conditioned medium, or extracellular matrix preparations.

What dilution should I start with for Western blot with this ADAMTS-6 antibody?

Start at 1:1000 dilution in your blocking buffer for Western blot, as recommended for this antibody. This concentration typically provides good signal-to-noise with overnight incubation at 4°C. If you are working with conditioned medium or samples with low ADAMTS-6 expression, you may need to adjust to 1:500. Always run a dilution series (1:500, 1:1000, 1:2000) with your specific sample type during initial optimization, as ADAMTS-6 expression levels vary considerably across cell lines and tissues. Extracellular matrix preparations often require more concentrated antibody than whole cell lysates.

Is this ADAMTS-6 antibody validated for immunohistochemistry?

Yes, Triple Point Biologics antibodies are validated for immunohistochemistry as part of our standard platform. For ADAMTS-6 IHC, expect predominantly extracellular matrix staining given the secreted, ECM-localized nature of this protease. Antigen retrieval is typically required; citrate buffer (pH 6.0) heat-induced epitope retrieval is a reasonable starting point. Because ADAMTS-6 expression can be focal and context-dependent, include appropriate positive control tissues. Lung adenocarcinoma samples have shown ADAMTS-6 expression in recent studies examining epithelial-mesenchymal transition, making them potentially useful positive controls.

Does this antibody cross-react with other ADAMTS family members?

The ADAMTS family comprises 19 members with conserved domain architecture, so cross-reactivity is a valid concern. This rabbit polyclonal was raised against ADAMTS-6-specific epitopes, but we recommend confirming specificity for your application. Run lysates from cells with known differential expression of ADAMTS family members, or use siRNA/CRISPR knockdown of ADAMTS6 as a negative control. If you observe unexpected bands or staining patterns, consider that some may represent ADAMTS-6 processing fragments rather than cross-reactivity. We have not systematically tested this antibody against all 18 other ADAMTS proteins.

Will this antibody work with dog samples?

Dog (canine) reactivity is predicted based on sequence homology between human and dog ADAMTS-6 orthologs, but has not been experimentally validated in our hands. The conserved regions show high identity, suggesting the antibody should recognize canine ADAMTS-6, but we recommend treating dog samples as experimental until you confirm detection. Run a known positive control (validated human sample) in parallel with your canine samples. If working with dog tissues, lung or cartilage may provide reasonable starting material given ADAMTS expression patterns, though specific canine ADAMTS-6 expression atlases remain limited.

Should I use cell lysate or conditioned medium to detect ADAMTS-6?

Because ADAMTS-6 is a secreted metalloprotease that localizes to the extracellular matrix, conditioned medium often yields stronger signal than whole cell lysates, particularly for cells actively secreting the protein. Concentrate conditioned medium 10-20 fold using centrifugal filters (30-50 kDa MWCO) before loading. For adherent cells, consider collecting the ECM fraction using established protocols, as mature ADAMTS-6 associates with matrix components. If using lysates, include both intracellular and membrane fractions. The choice depends on whether you are studying biosynthesis (lysate) versus secretion and ECM deposition (conditioned medium, ECM fraction).

What controls should I include when working with ADAMTS-6 antibody?

For Western blot, include a positive control cell line or tissue known to express ADAMTS-6. Lung adenocarcinoma cell lines undergoing EMT have documented ADAMTS-6 expression. A negative control using ADAMTS6 knockout or stable knockdown cells is ideal for confirming band identity. For IHC, run serial sections with and without primary antibody, and consider peptide competition if you have access to the immunogen sequence. Because ADAMTS-6 substrate specificity remains incompletely characterized, functional validation (protease activity assays, substrate degradation) can complement immunodetection when studying biological roles.

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

Store the antibody at -20°C in single-use aliquots to avoid repeated freeze-thaw cycles, which can compromise polyclonal antibody performance. The 100 µg pack size allows you to prepare multiple working aliquots. In our experience, properly stored aliquots maintain activity for at least two years from the manufacturing date. For short-term use (up to one month), diluted antibody can be stored at 4°C with 0.02 percent sodium azide as preservative. Avoid storing diluted antibody long-term, as this increases degradation risk. If you observe reduced signal over time, prepare a fresh aliquot rather than increasing concentration.