Anti-ACE-1 Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight should I expect for ACE-1 on a Western blot?

Somatic ACE (sACE), the predominant isoform in endothelial and epithelial tissues, runs at approximately 170 kDa due to extensive N-linked glycosylation; the deglycosylated polypeptide is closer to 150 kDa. Testicular ACE (tACE), expressed only in male germ cells, contains a single catalytic domain and migrates near 90–110 kDa depending on glycosylation state. A soluble cleaved form released by ACE secretase can appear around 160 kDa in serum and conditioned media. Run reducing SDS-PAGE on 7.5% or 4–15% gradient gels; higher-percentage gels compress the resolution in this MW range.

What is the recommended starting dilution for Western blot?

Start at 1:1000 in 5% non-fat milk or BSA in TBST, overnight at 4°C. For lysates with lower ACE expression (e.g., whole brain, non-endothelial cell lines), titrate down to 1:500. For high-expressing samples such as lung, kidney cortex, or HUVEC lysates, 1:2000–1:5000 is often sufficient. Load 20–40 µg total protein per lane. Because ACE is heavily glycosylated, avoid boiling samples above 70°C for more than 5 minutes — aggregation in the stacking gel is a common cause of signal loss at the expected MW.

Does this antibody distinguish somatic ACE from testicular ACE?

The immunogen spans a region present in both isoforms, so the antibody detects sACE and tACE. The two are easily distinguished by MW (~170 kDa versus ~100 kDa) and tissue distribution: tACE is restricted to post-meiotic male germ cells, while sACE dominates in vascular endothelium, kidney brush border, lung, and monocytes/macrophages. If you need isoform specificity, pair this antibody with a tACE-specific reagent raised against the unique N-terminal 67 residues of the testicular form, or use testis versus somatic tissue side-by-side as discriminating controls.

What positive control tissues or cell lines work well?

Human lung and kidney lysates are reliable high-expression controls for sACE. Among cell lines, HUVECs, HMEC-1, and Caco-2 (apical brush border) give strong signal. SW480 and HEK293 stably transfected with ACE constructs are useful for overexpression validation. For IHC, human kidney sections show characteristic brush border staining of proximal tubules and vascular endothelial labelling. Negative controls: ACE-knockdown HUVECs or lysates from cells pretreated with ACE-targeting siRNA. Captopril or lisinopril treatment does not reduce ACE protein levels and is not a suitable knockdown control.

Will this antibody cross-react with mouse or rat ACE?

Reactivity is validated in human. Mouse, rat, pig, dog, and Pan troglodytes reactivity is predicted based on sequence homology across the immunogen region (typically >85% identity for the mammalian orthologs listed), but has not been empirically confirmed by TPB. If you are working in mouse or rat, run a pilot blot against a known positive tissue (mouse lung or rat kidney) alongside human lung lysate before committing to a full experiment. Report back — confirmed cross-reactivity data from end users feeds into our validation database.

Can I use this antibody for immunoprecipitation or ACE activity assays?

The antibody is validated for Western blot. IP has not been formally validated, but rabbit polyclonals against extracellular ACE epitopes generally perform in native IP; expect to use 2–5 µg per mg of lysate and pre-clear thoroughly given the glycoprotein's tendency to bind non-specifically. For activity assays, immunocapture followed by fluorogenic substrate (e.g., Mca-RPPGFSAFK(Dnp)-OH or Hip-His-Leu) is feasible, but confirm the antibody does not occlude either catalytic domain by comparing captured versus total activity.

Why am I seeing multiple bands or a smear around 170 kDa?

ACE is heavily N-glycosylated at up to 17 sites, producing tissue-specific glycoforms that migrate as a doublet or diffuse band between 150 and 180 kDa. This is genuine signal, not degradation. Treatment with PNGase F collapses the bands to ~150 kDa and is a useful confirmation. Additional bands at ~90 kDa in testis samples represent tACE. Bands below 100 kDa in non-testis samples may indicate proteolytic cleavage during lysis — include 1 mM PMSF, EDTA-free protease inhibitors, and keep lysates cold. Avoid repeated freeze-thaw of membrane preparations.

How should I store the antibody and how stable is it?

Store at -20°C for long-term (up to 12 months from receipt) in the supplied buffer. For working stocks used within 4 weeks, 4°C is acceptable. Aliquot on first thaw into 5–10 µL volumes to avoid repeated freeze-thaw cycles, which degrade polyclonal performance over time. Do not store at -80°C unless you plan to thaw only once — the buffer is not optimized for ultra-low temperature cycling. If precipitation appears after thawing, centrifuge briefly at 10,000 × g and use the supernatant; activity is generally retained.