Anti-ADAM-21 Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight should I expect for ADAM-21 on a Western blot?

Full-length ADAM-21 (UniProt Q9UKJ8) comprises 722 amino acids with a predicted molecular weight near 80 kDa. On reducing SDS-PAGE, expect the mature glycosylated form between 85–95 kDa depending on post-translational modifications. ADAM family proteins undergo proteolytic processing; if you observe bands around 50–60 kDa, consider whether your sample contains the catalytic domain after prodomain cleavage. Testis lysates typically yield the full-length form. Always include a positive control lysate from testis tissue or transfected cells overexpressing ADAM-21 to confirm band identity.

What starting dilution should I use for ADAM-21 Western blots?

We recommend starting at 1:1000 dilution for Western blot, which has been validated with this polyclonal antibody. Testis lysates and cell lines with endogenous ADAM-21 expression typically produce clear signal at this dilution with overnight incubation at 4°C. If background is high, titrate up to 1:2000. For samples with low expression or to detect processed fragments, you may need to concentrate lysates or try 1:500. Detection sensitivity varies with ECL substrate choice; use a high-sensitivity reagent for low-abundance samples.

Is ADAM-21 expression limited to testis, or will I detect it in other tissues?

ADAM-21 shows predominant testis-specific expression in mammals, particularly in germ cells undergoing maturation. Low-level transcripts have been reported in epididymis and in certain epithelial cell types involved in ion transport, though protein detection outside reproductive tissues remains inconsistent. If screening non-reproductive tissues or cancer cell lines, prepare for weak or absent signal. Positive testis lysate controls are essential to confirm antibody performance. Some metastatic tumor models may show aberrant ADAM-21 upregulation; verify by RT-PCR if exploring oncology samples.

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

This ADAM-21 antibody is validated for human samples. Cross-reactivity with non-human primate and canine orthologs is predicted based on sequence homology in the immunogen region, but has not been empirically validated. Mouse and rat ADAM-21 share moderate sequence identity with human; we have not tested reactivity in these species. If working with rodent models, plan to include a known-positive human control and confirm band identity by peptide competition or knockout lysate. Pan-mammalian reactivity claims require independent verification for your specific model organism.

What sample preparation steps are critical for detecting ADAM-21?

ADAM-21 is a transmembrane metalloproteinase; use RIPA or a detergent-containing lysis buffer with protease inhibitors to solubilize membrane fractions effectively. For testis tissue, mechanical homogenization followed by brief sonication improves yield. Because ADAM proteins are susceptible to autocatalytic cleavage, keep samples on ice and add EDTA (5 mM) or a metalloproteinase inhibitor cocktail during lysis. Load 20–40 µg total protein per lane for endogenous detection. If purifying sperm, consider swim-up or gradient separation before lysis to enrich mature populations expressing ADAM-21.

Can I use this ADAM-21 antibody for immunohistochemistry on paraffin sections?

Triple Point antibodies are validated for Western blot; IHC application data is in progress. For ADAM-21, antigen retrieval is typically required; citrate buffer (pH 6.0) heat-mediated retrieval for 20 minutes is a standard starting condition. Testis sections should show specific staining in spermatocytes and round spermatids. Dilution range for IHC is generally 1:100 to 1:200, though optimal concentration depends on fixation time and tissue type. Include a negative control (non-immune rabbit IgG) and confirm localization matches expected germ-cell or membrane patterns reported in the literature.

How should I store the ADAM-21 antibody, and what is the 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. The 100 µg pack size is supplied in a stabilized buffer; upon receipt, spin briefly to collect contents, then aliquot 10–20 µL portions for typical experimental use. Stability at –20°C is at least 12 months from manufacture date when stored properly. For short-term use (up to one month), diluted working stock can be held at 4°C with 0.02% sodium azide, though we recommend preparing fresh dilutions for best results.

Are there known ADAM-21 isoforms or splice variants I should be aware of?

The primary ADAM-21 transcript encodes the 722-amino acid reference isoform (UniProt Q9UKJ8-1). Alternative splicing has been reported in testis cDNA libraries, though functional characterization of variants remains limited. Most variants involve truncations in the cytoplasmic tail or alterations in the cysteine-rich domain, which may not substantially alter recognition by an antibody raised against the extracellular catalytic and disintegrin domains. If you observe multiple bands on Western blots, consider splice variants, proteolytic processing, or glycosylation heterogeneity. Peptide competition assays can help distinguish specific signal from cross-reactive species.