Anti-ADAMTS-8 Rabbit Polyclonal Antibody

Frequently Asked Questions

What is the expected molecular weight for ADAMTS-8 on Western blot?

Full-length ADAMTS-8 has a predicted molecular weight of approximately 103 kDa based on its 889-amino acid sequence. However, as a secreted protease with extensive post-translational modifications including glycosylation, the apparent molecular weight on SDS-PAGE typically ranges from 110-120 kDa. You may also detect processed forms or fragments depending on sample type, since ADAMTS-8 undergoes proteolytic maturation after secretion. Conditioned media from cells expressing ADAMTS-8 often show multiple bands corresponding to the proenzyme, mature enzyme, and cleavage products. For initial experiments, expect the major band between 110-120 kDa in cell lysates and conditioned medium.

What starting dilution should I use for ADAMTS-8 Western blot?

We recommend starting at 1:1000 for Western blot with overnight incubation at 4°C. This dilution has been validated with human samples. Because ADAMTS-8 is a secreted protein, signal intensity varies significantly between cell lysates and conditioned media. Conditioned medium often requires less antibody; you may achieve good signal at 1:2000. If working with tissue lysates where ADAMTS-8 expression is low, start at 1:500 and titrate upward. Always include a positive control such as conditioned medium from HT1080 or A549 cells, which express detectable endogenous ADAMTS-8.

Does this antibody work for mouse and rat ADAMTS-8?

The antibody is validated for human ADAMTS-8. Reactivity with mouse and rat is predicted based on sequence homology but not experimentally validated by Triple Point Biologics. Human and mouse ADAMTS-8 share approximately 75% identity across the full protein, with higher conservation in the catalytic domain. If working with rodent samples, plan to empirically test the antibody starting at the recommended 1:1000 dilution. Include a human positive control in parallel. Researchers have reported variable success with cross-species detection of ADAMTS family members, so pilot experiments are essential before committing to large-scale studies.

Should I use conditioned medium or cell lysate for detecting ADAMTS-8?

Because ADAMTS-8 is a secreted metalloprotease, conditioned medium is generally the preferred sample type and provides stronger, cleaner signal than cell lysate. Cells secrete ADAMTS-8 into the extracellular space where it associates with matrix components. To prepare conditioned medium, culture cells in serum-free or low-serum medium for 24-48 hours, collect supernatant, clarify by centrifugation, and concentrate 10-20-fold using centrifugal filters (10 kDa cutoff). Whole-cell lysates contain primarily intracellular proenzyme. For adherent cells producing extracellular matrix, consider extracting matrix-associated ADAMTS-8 using chaotropic agents after removing cells.

Can this antibody distinguish between pro-ADAMTS-8 and the mature enzyme?

This polyclonal antibody recognizes epitopes across ADAMTS-8 and will detect both the proenzyme and mature, processed forms. It does not selectively recognize only the pro-domain or only the mature catalytic domain. On Western blot you may observe multiple bands representing the full-length proenzyme, the mature enzyme after pro-domain removal, and further proteolytic fragments. The banding pattern provides information about processing state but the antibody itself does not discriminate between forms. If you need to specifically detect activation state, consider combining this antibody with functional assays or using samples treated with protease inhibitors to preserve the proenzyme.

What positive and negative controls should I use for ADAMTS-8 experiments?

For positive controls, use conditioned medium from cell lines with documented ADAMTS-8 expression such as HT1080 fibrosarcoma or A549 lung carcinoma cells. Alternatively, recombinant human ADAMTS-8 protein serves as a defined positive control for validating antibody performance and confirming expected molecular weight. For negative controls, use medium alone or conditioned medium from cells with negligible ADAMTS-8 expression. Cell lines treated with the translation inhibitor cycloheximide provide additional specificity controls. Include molecular weight markers spanning 75-150 kDa. For IHC, normal tissues with low ADAMTS-8 expression (such as normal liver) serve as useful negative references.

Are there known ADAMTS-8 isoforms I should be aware of?

The primary annotated isoform of ADAMTS-8 (UniProt Q9UP79-1) is 889 amino acids. Alternative splicing has been reported but validated functional isoforms are not well-characterized in the literature compared to other ADAMTS family members. Most published work focuses on the full-length secreted form. You may observe multiple bands on Western blot, but these typically represent post-translational processing (furin cleavage of the pro-domain, glycosylation heterogeneity, or extracellular proteolytic cleavage) rather than splice variants. If working with tissue samples or complex biological fluids, expect heterogeneous banding due to processing rather than true isoform diversity.

How should I store this ADAMTS-8 antibody and what is the shelf life?

Store the antibody at -20°C in small aliquots to avoid repeated freeze-thaw cycles, which can reduce titer and increase background. The antibody is supplied as a rabbit polyclonal in a buffered solution, typically stable for at least 12 months at -20°C. Once thawed, an aliquot can be stored at 4°C for up to one month for convenience during active experiments. Do not store diluted antibody; prepare working dilutions fresh in blocking buffer immediately before use. If you observe increased background or reduced signal over time, this often indicates degradation from repeated temperature cycling rather than expiration of properly stored stock.