Anti-ADAMTS-1 Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight band should I expect for ADAMTS-1 on Western blot?

Full-length ADAMTS-1 has a predicted molecular weight of approximately 110 kDa based on its 967-amino acid sequence. However, ADAMTS-1 undergoes post-translational processing, including signal peptide cleavage and potential proteolytic maturation, so you may observe the mature form around 87-100 kDa. Glycosylation can shift the apparent size upward. Some researchers also report lower molecular weight bands representing proteolytic fragments or processing intermediates. Running a positive control lysate from cells known to secrete ADAMTS-1, such as activated fibroblasts or certain ovarian cell lines, will help you establish the expected pattern in your system.

What starting dilution should I use for Western blot with this ADAMTS-1 antibody?

We recommend starting at 1:1000 dilution for Western blot applications. This rabbit polyclonal has been validated at that working concentration. Depending on your sample's ADAMTS-1 expression level and your detection system sensitivity, you may need to optimize between 1:500 and 1:2000. Because ADAMTS-1 is a secreted protein, conditioned media often yields stronger signal than cell lysates. If working with tissue lysates or low-expressing cells, consider concentrating your sample or starting at the higher antibody concentration. Include a titration series in your first experiment to identify the dilution that minimizes background while preserving signal.

Is this antibody likely to work in mouse samples even though only human is validated?

Mouse is listed as potential cross-reactivity based on sequence homology between human and mouse ADAMTS-1, which share approximately 84% amino acid identity. The antibody has not been formally validated in mouse samples by us, so you should treat initial experiments as optimization runs. Include a known positive control, such as lysate from mouse ovarian tissue or TNF-α-stimulated mouse fibroblasts, which express ADAMTS-1 endogenously. If you observe bands at unexpected sizes or high background, the cross-reactivity may be lower than predicted. Peptide competition or siRNA knockdown controls will help confirm specificity in the mouse system.

Should I use reducing or non-reducing conditions when running ADAMTS-1 samples?

Use reducing conditions with beta-mercaptoethanol or DTT. ADAMTS-1 contains multiple disulfide bonds within its thrombospondin type-1 repeats and other domains, but Western blot detection typically requires denaturation and reduction to expose epitopes and achieve the expected monomer migration. Non-reducing conditions may result in higher molecular weight species due to disulfide-linked aggregates or retention of compact tertiary structure, complicating interpretation. If you are specifically investigating disulfide-dependent complexes or ADAMTS-1 interactions with ECM components, run parallel reduced and non-reduced gels, but for routine detection of ADAMTS-1 protein, reducing conditions are standard.

Can I detect ADAMTS-1 in conditioned media or only in cell lysates?

ADAMTS-1 is a secreted protein, so conditioned media is often the preferred sample type and typically yields stronger signal than cell lysates. After culturing cells under serum-free or low-serum conditions for 24-48 hours, concentrate the conditioned media 10- to 20-fold using centrifugal filter units with a 10 kDa cutoff before loading onto gels. This concentrates secreted ADAMTS-1 while removing albumin and other high-abundance serum proteins that may interfere. Cell lysates can be used but may show lower signal since much of the protein is secreted. Heparin-affinity enrichment can further enhance detection, as ADAMTS-1 binds heparin through its spacer domain.

Does ADAMTS-1 have isoforms or splice variants I should be aware of?

The primary reference sequence for human ADAMTS-1 is the full-length 967-amino acid isoform. Alternative splicing has not been extensively documented for ADAMTS-1 compared to some other ADAMTS family members. However, ADAMTS-1 does undergo proteolytic processing after secretion, with cleavage events that can generate N-terminal or C-terminal fragments retaining catalytic or ancillary domain activity. Depending on the epitope recognized by this antibody, you may detect the full-length proenzyme, the mature processed form, or specific fragments. The antibody target region is not specified here, so if you observe multiple bands, consider that they may represent processing intermediates rather than splice variants.

What positive and negative controls should I include when using this ADAMTS-1 antibody?

For a positive control, use lysate or conditioned media from cells with known ADAMTS-1 expression, such as human foreskin fibroblasts stimulated with TNF-α or IL-1β, which upregulate ADAMTS-1 transcription. Human granulosa-lutein cells or certain ovarian cancer cell lines also express detectable levels. For a negative control, consider using lysate from cells with minimal ADAMTS-1 expression, or perform siRNA-mediated knockdown of ADAMTS-1 in your experimental cell line and compare to scrambled siRNA. Peptide competition with the immunizing peptide, if available, provides additional specificity confirmation. Recombinant ADAMTS-1 protein can serve as a positive standard but may migrate differently than the endogenous protein.

How should I store this 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 titer and increase aggregation. Rabbit polyclonal antibodies in glycerol-containing buffers are generally stable for at least two years at -20°C under these conditions. For short-term use over several weeks, an aliquot can be kept at 4°C with sodium azide as preservative if your application tolerates it; do not use azide-containing antibody for immunofluorescence on live cells or HRP-based detection. Avoid storing diluted antibody; prepare working dilutions fresh in blocking buffer on the day of use. Centrifuge briefly before use if you observe any precipitate after thawing.