Anti-ADAMTS-14 Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight should I expect for ADAMTS-14 on Western blot?

ADAMTS-14 (UniProt Q8WXS8) has a calculated molecular weight of approximately 134 kDa for the full-length 1,223-amino acid protein. However, you will likely observe the protein running between 140-160 kDa due to extensive glycosylation. ADAMTS-14 is synthesized as a latent zymogen and requires proteolytic activation to display enzymatic activity, which may generate lower molecular weight processed forms. If you detect multiple bands, consider that the enzyme undergoes post-translational processing and that different activation states or proteolytic fragments may be present depending on your sample type and extraction conditions.

What dilution should I start with for Western blot detection of ADAMTS-14?

We recommend starting at 1:1,000 dilution for Western blot, which has been validated with this antibody. Because ADAMTS-14 is a secreted extracellular matrix protein, expression levels vary considerably by cell type and culture conditions. Fibroblasts and cells actively remodeling collagen typically show higher expression. If your initial signal is weak, titrate up to 1:500. For samples with low endogenous expression, consider enriching ECM proteins during lysis or concentrating conditioned media before running your gel. Always include a positive control lysate from cells known to express ADAMTS-14, such as dermal fibroblasts.

Does this antibody cross-react with mouse or rat ADAMTS-14?

Human ADAMTS-14 has been validated as the primary species reactivity for this rabbit polyclonal antibody. Mouse and rat cross-reactivity is predicted based on sequence homology but has not been experimentally confirmed in our hands. Human and mouse ADAMTS-14 share approximately 85% amino acid identity, which suggests reasonable cross-reactivity likelihood. If you are working with rodent samples, we recommend testing the antibody at the standard 1:1,000 dilution first, but be prepared to optimize or include a positive human control for comparison. Confirm any observed bands by molecular weight and known expression patterns in your tissue or cell type.

Why am I not detecting ADAMTS-14 in my cell lysates?

ADAMTS-14 is a secreted metalloproteinase that localizes to the extracellular matrix, so whole-cell lysates often show weak or absent signal. The protein is secreted into conditioned medium or deposited into the ECM rather than retained intracellularly. To improve detection, collect conditioned culture medium after 24-48 hours, concentrate it by ultrafiltration or TCA precipitation, and probe that material. Alternatively, extract ECM-associated proteins using urea or chaotropic buffers after removing cells. Intracellular detection typically captures only nascent or trafficking protein. Fibroblasts and stromal cells generally express higher levels than epithelial lines. Verify your positive control expresses ADAMTS-14 under your culture conditions.

Can I use this ADAMTS-14 antibody for immunohistochemistry?

Yes, Triple Point Biologics antibodies are typically validated for immunohistochemistry in addition to Western blot. ADAMTS-14 is an extracellular matrix protein, so you should expect stromal, pericellular, and ECM staining patterns rather than intracellular localization. Start with a 1:100 to 1:200 dilution for IHC and optimize based on your tissue type and fixation protocol. Antigen retrieval is generally required; citrate buffer (pH 6.0) heat-mediated retrieval is a reasonable starting point. Include appropriate negative controls (primary antibody omission) and verify staining patterns match known ADAMTS-14 expression, particularly in connective tissue and fibroblast-rich regions.

What is the best positive control tissue or cell line for ADAMTS-14?

Dermal fibroblasts are a reliable positive control for ADAMTS-14, as these cells actively synthesize and secrete the enzyme for collagen processing in the extracellular matrix. Other collagen-rich tissues such as skin, tendon, and ligament typically show detectable expression. Given recent implication of ADAMTS-14 in colorectal cancer, certain colorectal cancer cell lines or tumor lysates may also serve as positive controls, though expression is context-dependent. When using cell culture models, collect conditioned medium or ECM fractions rather than whole-cell lysates, since ADAMTS-14 is secreted. Confirm expression in your chosen control by checking published transcriptomic or proteomic datasets before committing to large-scale experiments.

How should I store this ADAMTS-14 antibody and what is the expected shelf life?

Store the antibody at -20°C in single-use aliquots to avoid repeated freeze-thaw cycles, which can reduce titre and increase background. The 100 µg pack size is sufficient for aliquoting into 10-20 µL volumes depending on your expected usage. Polyclonal antibodies are generally stable for 12-24 months under these conditions. Avoid storing in frost-free freezers where temperature cycling occurs. Once thawed, an aliquot can be held at 4°C for up to one month if supplemented with 0.02% sodium azide or another preservative. Do not store diluted antibody in working solution for extended periods; prepare fresh dilutions in blocking buffer for each experiment to maintain optimal performance.

Does ADAMTS-14 require activation to be detected, and will this antibody recognize both pro- and active forms?

ADAMTS-14 is synthesized as an inactive zymogen and requires proteolytic cleavage of its propeptide to become enzymatically active. This rabbit polyclonal was raised against a region spanning residues within the mature protein, so it should recognize both the latent pro-form and the activated enzyme, though the exact epitope has not been mapped in our hands. On Western blot, you may observe multiple bands corresponding to the zymogen, activated form, and processing intermediates depending on your sample. The presence of both forms is biologically relevant, as activation state varies by tissue context and the presence of activating proteases. Band pattern will depend on whether your sample contains predominantly latent or processed enzyme.