Anti-ADAM-9 Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight should I expect for ADAM-9 on Western blot?

Full-length ADAM-9 migrates at approximately 90-110 kDa on Western blot, reflecting the 819-residue protein with post-translational glycosylation. You may also detect bands around 70-80 kDa corresponding to the mature, processed form after prodomain cleavage. Some lysates show additional lower bands representing shed ectodomains or C-terminal fragments, particularly in samples from highly proteolytic environments like tumor cell lines. Running a positive control lysate from ADAM-9-expressing cells alongside molecular weight markers will help distinguish the full-length species from processing intermediates in your specific system.

Does this antibody recognize both membrane-bound and shed forms of ADAM-9?

The epitope region will determine which forms are detected. Because ADAM-9 undergoes ectodomain shedding at the cell surface, antibodies targeting the extracellular domain may recognize both membrane-anchored and soluble shed fragments, while those directed at the cytoplasmic tail detect only the membrane-tethered form. If you are analyzing conditioned media or serum for soluble ADAM-9, confirm the epitope falls within the ectodomain. For total cellular ADAM-9, whole-cell lysates prepared with detergent will capture both membrane-bound and any intracellular pools.

What is the recommended starting dilution for ADAM-9 Western blot?

Start at 1:1000 dilution for Western blot, which typically provides good signal-to-noise with overnight incubation at 4°C. If ADAM-9 expression is low in your cell type—common in non-cancerous or epithelial lines—you may need to concentrate to 1:500 or load more total protein per lane. Conversely, in myeloma, glioblastoma, or other ADAM-9-overexpressing models, 1:2000 may suffice. Always include a lysate control known to express ADAM-9 during your first titration to establish linearity before comparing experimental samples.

Is this ADAM-9 antibody validated for mouse samples or only human?

This antibody is validated in human samples. Mouse cross-reactivity is predicted based on sequence homology between human and mouse ADAM-9, which share approximately 85% identity in conserved domains, but we have not empirically confirmed detection in mouse lysates. If you are working with mouse tissue or cell lines, plan to run a positive control—such as lysate from mouse macrophages or endothelium known to express ADAM-9—and verify band specificity. Primate and canine samples show higher predicted cross-reactivity due to closer sequence conservation.

What sample preparation method works best for detecting ADAM-9?

Because ADAM-9 is a membrane-anchored metalloproteinase, use RIPA or a similar detergent-containing lysis buffer to solubilize the protein effectively. Avoid prolonged incubation at room temperature during lysis, as ADAM-9 retains catalytic activity and can autodegraded or cleave substrates in the lysate. Include protease inhibitors—particularly metalloproteinase inhibitors like EDTA or 1,10-phenanthroline—to preserve full-length ADAM-9. For IHC, antigen retrieval with citrate buffer pH 6.0 typically improves epitope accessibility in formalin-fixed paraffin-embedded sections.

What cell lines or tissues serve as good positive controls for ADAM-9?

ADAM-9 is robustly expressed in myeloma cell lines such as U266 and RPMI-8226, reflecting its original identification in myeloma. Glioblastoma lines, including U87-MG, and several breast cancer models also show high endogenous expression. Normal human placenta and lung tissue are reliable positive controls for IHC, as ADAM-9 is expressed in trophoblasts and bronchial epithelium. For negative controls, consider tissues or cell lines with minimal ADAM-9 transcript, though true negatives are uncommon given its broad, low-level expression across many cell types.

Can this antibody distinguish between active and inactive ADAM-9?

Standard antibodies, including this polyclonal, recognize ADAM-9 regardless of activation state and cannot discriminate between the inactive proform and the mature, catalytically active enzyme by immunoblot or IHC alone. The proform contains an N-terminal prodomain that is cleaved by furin-like convertases to generate the active species, but both migrate closely on SDS-PAGE after reduction. If you need to assess ADAM-9 activity specifically, pair immunodetection with a functional assay such as fluorogenic peptide cleavage or analyze known activation markers in your biological context.

How should I store the ADAM-9 antibody to maintain long-term stability?

Store the antibody at -20°C in small aliquots to avoid repeated freeze-thaw cycles, which can reduce titer and increase aggregation. If you use the antibody frequently, a working aliquot can be held at 4°C for up to one month with 0.02% sodium azide as preservative, though this is not compatible with applications requiring live cells or HRP-conjugated detection systems sensitive to azide inhibition. Do not store diluted antibody in blocking buffer for more than a week, as carrier proteins can degrade and promote bacterial growth.