Anti-ADAMTS-12 Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight band should I expect for ADAMTS-12 on Western blot?

Full-length human ADAMTS-12 (UniProt P58397) is 1594 amino acids with a predicted molecular weight around 175-180 kDa. On reducing SDS-PAGE, expect the primary band near 180 kDa, though migration can vary depending on glycosylation status. ADAMTS-12 is a secreted metalloprotease that undergoes post-translational modifications in the extracellular space, so you may observe additional bands representing processed forms or glycosylation variants, particularly in conditioned media samples. Use positive control lysates from tissues or cells known to express ADAMTS-12, such as cartilage extracts or appropriate cancer cell lines.

What starting dilution should I use for ADAMTS-12 Western blot?

Start at 1:1000 dilution in blocking buffer for Western blot, which falls within our validated range. This concentration works well for cell lysates and conditioned media with moderate ADAMTS-12 expression. If signal is weak, titrate upward to 1:500. If background is high, extend to 1:2000. Because ADAMTS-12 is secreted and often present at lower abundance than cytosolic proteins, concentrated conditioned media or tissue extracts from cartilage, tendon, or stromal compartments typically yield stronger signal than whole-cell lysates. Include appropriate loading controls to normalize for sample input.

Is this ADAMTS-12 antibody validated for mouse and rat samples?

The antibody is validated for human ADAMTS-12. Mouse and rat cross-reactivity is predicted based on sequence homology in the epitope region but not experimentally confirmed by us. Human and mouse ADAMTS-12 share approximately 85% overall identity, with higher conservation in the catalytic and ancillary domains. If working with rodent samples, plan to run positive controls and verify band specificity. Researchers have reported success using human-reactive ADAMTS antibodies in mouse cartilage and tumour models, but you should validate performance in your specific model and tissue type before committing to large-scale experiments.

Does ADAMTS-12 require special sample preparation or lysis conditions?

Because ADAMTS-12 is secreted into the extracellular matrix, sample preparation depends on your experimental context. For conditioned media, concentrate 10- to 20-fold using centrifugal filters (10 kDa cutoff) before loading. For tissue samples rich in ECM (cartilage, tendon, tumour stroma), include a brief protease inhibitor cocktail and consider sequential extraction: first a mild detergent lysis, then a higher-stringency buffer to solubilize matrix-embedded protein. Avoid prolonged boiling, which can aggregate heavily glycosylated proteases. Reducing conditions (DTT or β-mercaptoethanol) are standard; non-reducing gels may show altered migration due to disulfide bonds in the thrombospondin domains.

Can I use this antibody for immunohistochemistry on paraffin sections?

Triple Point Biologics antibodies are validated for Western blot; IHC application data is in progress, and this polyclonal format is generally suitable for paraffin-embedded tissue. ADAMTS-12 localizes to the extracellular matrix, so expect staining in stromal compartments, pericellular regions in cartilage, and tumour microenvironments depending on tissue type. Antigen retrieval is typically required; citrate buffer (pH 6.0) heat-induced epitope retrieval is a reasonable starting point. Optimize antibody dilution empirically, beginning around 1:100 to 1:200. Include positive-control sections from tissues with known ADAMTS-12 expression (e.g., growth-plate cartilage, certain carcinomas) and negative controls (omit primary or use isotype control) to confirm specificity.

What are good positive and negative controls for ADAMTS-12 experiments?

Positive controls depend on your model. Human chondrocytes, osteosarcoma cell lines (e.g., U2OS), and certain breast or colorectal cancer lines express detectable ADAMTS-12. Conditioned media from these cells often yield cleaner signal than whole lysates. For tissue, articular cartilage and tendon are physiological sources. Negative controls should include lysates from cells with low or absent ADAMTS-12 expression; verify by qPCR if uncertain. Additionally, pre-incubating the antibody with recombinant ADAMTS-12 peptide (if available) or using siRNA/CRISPR knockdown samples provides specificity confirmation. Always include a no-primary-antibody control lane to assess non-specific secondary binding.

How should I store the ADAMTS-12 antibody and what is its shelf life?

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 suitable for dividing into 10-20 µL working aliquots depending on your experimental frequency. Once thawed, an aliquot can be kept at 4°C for up to one month if sodium azide is present as preservative (check the product datasheet). For long-term stability, keep aliquots at –20°C or –80°C. Polyclonal antibodies from rabbit sera typically remain stable for two to three years under these conditions. Avoid contamination by using sterile technique when removing aliquots.

Will this antibody detect ADAMTS-12 in cell culture supernatants or only in lysates?

This antibody is well suited for detecting ADAMTS-12 in conditioned media, which is often the preferred sample type since ADAMTS-12 is secreted. Collect serum-free or low-serum conditioned media after 24-48 hours of culture, then concentrate 10- to 20-fold to enrich for secreted proteases. Load equivalent cell numbers or normalize by total secreted protein (e.g., BCA assay on unconcentrated media). ADAMTS-12 can also be detected in cell lysates, particularly if cells are harvested during active secretion, but intracellular pools are smaller. Comparing lysate and media side-by-side helps assess secretion efficiency and potential retention in the secretory pathway or cell-surface anchorage.