Anti-ADAM-12 Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight band should I expect for ADAM-12 on Western blot?

Full-length ADAM-12 runs at approximately 90-120 kDa on Western blot, though the exact mobility depends on glycosylation status and which isoform is expressed. ADAM-12L (the long, membrane-bound form) typically appears around 120 kDa, while ADAM-12S (the short, secreted isoform lacking the transmembrane domain) migrates closer to 90 kDa. If you are working with cell lysates that secrete ADAM-12, you may detect both forms. Post-translational modifications, particularly N-glycosylation within the disintegrin and cysteine-rich domains, can cause additional heterogeneity in migration.

What starting dilution should I use for ADAM-12 Western blot?

We recommend starting at 1:1000 for Western blot, which has been validated in-house with human lysates. Depending on ADAM-12 expression levels in your specific cell type or tissue, you may need to adjust between 1:500 and 1:2000. Muscle tissues and activated fibroblasts generally express ADAM-12 at detectable levels with overnight incubation at 4°C. If you are working with low-abundance samples or investigating ADAM-12 upregulation under hypoxic or inflammatory conditions, starting at 1:500 may improve signal without substantially increasing background.

Is this ADAM-12 antibody validated in mouse samples?

Human reactivity has been validated; mouse reactivity is predicted based on sequence homology but has not been formally validated by Triple Point Biologics. Human and mouse ADAM-12 share approximately 85 percent identity across the metalloproteinase and disintegrin domains, which are common epitope regions for polyclonal antibodies. We recommend running a positive control (human lysate or recombinant ADAM-12) in parallel with your mouse samples on the first blot. If you observe specific bands at the expected molecular weight in mouse, the cross-reactivity is likely sufficient for your application.

What are good positive and negative control cell lines for ADAM-12 expression?

Skeletal muscle myoblasts (such as human primary myoblasts or C2C12 mouse myoblasts during differentiation) are reliable positive controls, as ADAM-12 is upregulated during fusion. Many carcinoma lines, including MDA-MB-231 breast cancer cells, also express ADAM-12 at detectable levels, particularly under hypoxic culture conditions. For a negative control, consider using non-transformed epithelial lines with low basal metalloproteinase activity or ADAM-12 knockout cells if available. Comparing lysates from proliferating versus differentiating myoblasts can also serve as an internal dynamic-range control.

Can I use this antibody for immunofluorescence on fixed muscle sections?

Yes, Triple Point Biologics antibodies are validated for immunofluorescence and immunohistochemistry applications. For skeletal muscle cryosections, we suggest starting at a dilution between 1:100 and 1:200 after fixation in four percent paraformaldehyde or cold methanol. ADAM-12 localizes to the plasma membrane in myoblasts and at fusion sites, so you should observe peripheral staining in differentiating cultures. Antigen retrieval is typically not required for frozen sections, but if you are working with paraffin-embedded tissue, citrate buffer retrieval (pH 6.0) often improves signal.

Does this antibody distinguish between ADAM-12L and ADAM-12S isoforms?

This polyclonal antibody recognizes epitopes common to both the long membrane-anchored (ADAM-12L) and short secreted (ADAM-12S) isoforms, so it will not selectively detect one over the other by Western blot. The two isoforms differ in molecular weight (approximately 120 kDa versus 90 kDa), allowing you to infer which form is present based on band position. If your experimental question requires isoform-specific detection, consider fractionating cell lysates into membrane and secreted fractions, or use isoform-selective primers to confirm mRNA expression in parallel.

How should I prepare cell lysates to preserve ADAM-12 detection?

Use a lysis buffer containing protease inhibitors, particularly metalloproteinase inhibitors such as EDTA or 1,10-phenanthroline, to prevent autocatalytic cleavage or degradation by other matrix metalloproteinases in your lysate. RIPA buffer supplemented with protease inhibitor cocktail works well for total cell lysates. If you are specifically interested in the membrane-associated ADAM-12L isoform, include a non-ionic detergent such as one percent Triton X-100 to solubilize membrane proteins efficiently. Avoid repeated freeze-thaw cycles, which can lead to aggregation or proteolytic degradation of high-molecular-weight metalloproteinases.

What is the recommended storage condition and shelf life for this antibody?

Store the antibody at –20°C in single-use aliquots to avoid repeated freeze-thaw cycles, which can reduce titer and increase aggregation. The 100 µg pack size is supplied in a glycerol-containing buffer that remains stable for at least 12 months when stored properly. For long-term storage beyond one year, keep aliquots at –80°C. Once thawed, an aliquot can be held at 4°C for up to one month if sodium azide is present as a preservative. Always centrifuge briefly before use to pellet any aggregates that may have formed during storage.