Anti-ADAMTS-18 Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight should I expect for ADAMTS-18 on Western blot?

Full-length ADAMTS-18 has a predicted molecular weight of approximately 135 kDa based on its 1,221-amino acid sequence. However, the mature secreted form runs closer to 130 kDa after signal peptide cleavage. You may observe additional bands representing proteolytically processed fragments, particularly in conditioned media samples, since ADAMTS-18 undergoes autocatalytic or exogenous cleavage in the extracellular space. If working with intracellular lysates, expect lower abundance compared to secreted fractions. Running a positive control from tissue or cells known to express ADAMTS-18 (e.g., ocular tissue lysates) will help confirm the expected pattern.

Is this ADAMTS-18 antibody validated in mouse and rat samples?

The antibody is validated in human samples and shows predicted cross-reactivity with mouse, rat, dog, and non-human primate based on epitope sequence homology. We have not independently confirmed detection in rodent lysates. Human and mouse ADAMTS-18 share approximately 80–85% sequence identity in conserved domains, so cross-reactivity is likely but band intensity may differ. If working with mouse or rat, start at the recommended 1:1000 dilution and be prepared to optimize. Include a human positive control on the same blot to verify antibody performance before troubleshooting species-specific detection issues.

What starting dilution should I use for Western blot with this antibody?

Start at 1:1000 dilution for Western blot in 5% non-fat milk or BSA in TBS-T. ADAMTS-18 is a secreted extracellular matrix protein, so signal intensity depends heavily on sample type. Conditioned media from cultured cells typically yields stronger signal than whole-cell lysates. If background is high at 1:1000, increase to 1:2000 or switch blocking agent. For very low-abundance samples or if detecting endogenous protein in primary tissue, you may need to concentrate your sample or test 1:500. Overnight incubation at 4°C often improves sensitivity compared to one hour at room temperature for this target.

Can I use this antibody for immunofluorescence on fixed cells?

Yes, Triple Point Biologics antibodies are validated for immunofluorescence applications. Because ADAMTS-18 is secreted and associates with the extracellular matrix, you will see predominantly extracellular or pericellular staining rather than intracellular localization in most cell types. Use methanol or paraformaldehyde fixation; permeabilization is less critical since the target is extracellular. Start with a 1:100–1:200 dilution and include cells cultured in serum-free media for 24–48 hours to enrich secreted ADAMTS-18 in the matrix. A no-primary-antibody control is essential to distinguish specific ECM staining from non-specific matrix binding.

What is the best sample type for detecting endogenous ADAMTS-18?

Conditioned serum-free media from cultured cells or tissue lysates enriched for extracellular matrix proteins yield the strongest signal. ADAMTS-18 is secreted, so whole-cell lysates contain primarily intracellular precursor and lower total abundance. If using cultured cells, switch to serum-free medium for 24–48 hours before harvest to reduce serum protein interference and enrich secreted proteinases. For tissues, ocular tissues (cornea, retina) and cartilage show relatively high endogenous expression. Concentrate conditioned media by ultrafiltration (10 kDa cutoff) or TCA precipitation if initial signal is weak. Avoid freeze-thaw cycles, as ADAMTS-18 activity and epitope integrity may decline.

Does ADAMTS-18 have splice isoforms I should know about?

ADAMTS-18 has limited documented splice variation compared to some other ADAMTS family members. The major transcript encodes the full 1,221-amino acid protein. Shorter variants have been reported in transcript databases but are not well characterized at the protein level. On Western blot, multiple bands most often reflect proteolytic processing rather than isoforms, since ADAMTS-18 is subject to cleavage in the extracellular space. If you observe multiple bands, consider whether they represent N-terminal fragments retaining the metalloproteinase domain or C-terminal fragments with thrombospondin repeats. Epitope mapping information for this antibody can help predict which fragments will be detected.

What positive and negative controls should I use for ADAMTS-18 Western blot?

A validated positive control is conditioned medium from HEK293 or other cells transfected with ADAMTS-18 expression vector, or lysate from human tissue known to express the protein (ocular tissues, certain carcinoma lines). Negative controls include lysate from cells with confirmed low or absent ADAMTS-18 expression, or the same sample pre-incubated with blocking peptide if available. Because ADAMTS-18 is secreted, side-by-side comparison of cell lysate versus conditioned medium from the same culture provides an internal control showing enrichment in the secreted fraction. Omitting primary antibody confirms specificity of the secondary detection system.

How should I store this antibody and how long is it stable?

Store at -20°C as supplied. Rabbit polyclonal antibodies in glycerol-containing buffer are stable for at least 12 months at -20°C. Avoid repeated freeze-thaw cycles; aliquot into single-use volumes if you will use the antibody infrequently. For short-term use (up to one month), you may store a working aliquot at 4°C with 0.02–0.05% sodium azide as preservative. Do not store diluted antibody in blocking buffer for more than one week, as carrier proteins can degrade and background may increase. If you observe decreased signal or increased background over time, prepare a fresh aliquot from the -20°C stock.