Anti-ADAM-20 Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight should I expect for ADAM-20 on Western blot?

Full-length ADAM-20 (UniProt O43506) has a calculated molecular weight of approximately 84 kDa based on its 726-residue sequence. On reducing SDS-PAGE, expect the mature protein to migrate between 80-90 kDa depending on glycosylation status, as ADAM-20 contains multiple potential N-glycosylation sites in its extracellular domains. The proprotein form, if present in immature testicular lysates, may run slightly higher. Post-translational cleavage of the prodomain during sperm maturation can yield processed forms in the 70-75 kDa range. Run positive control lysates from human testis alongside your samples.

What starting dilution should I use for Western blot with this ADAM-20 antibody?

Start at 1:1000 dilution in blocking buffer for Western blot, the validated concentration for this rabbit polyclonal. ADAM-20 expression is restricted primarily to testicular tissue and mature spermatozoa, so signal intensity will depend heavily on your sample source. For whole testis lysates, 1:1000 typically provides good signal-to-noise. If working with isolated sperm fractions or low-abundance samples, you may need to titrate down to 1:500. Always include a testis positive control. Overnight incubation at 4°C often improves detection compared to one-hour room-temperature binding for membrane proteins like ADAM-20.

Is this ADAM-20 antibody likely to cross-react with mouse or rat samples?

Mouse and rat cross-reactivity are listed as potential but not yet validated for this antibody. Human ADAM-20 shares approximately 75-80 percent sequence identity with mouse and rat orthologs, concentrated in the metalloproteinase and disintegrin domains. If you plan to use rodent samples, treat this as experimental: run human testis lysate as a positive control alongside your mouse or rat samples at the same dilution, compare band patterns, and verify the expected molecular weight. Consider peptide competition or siRNA knockdown in a testable cell system to confirm specificity before committing to large-scale rodent experiments.

What sample preparation works best for detecting ADAM-20 in sperm lysates?

ADAM-20 is a transmembrane protein anchored in the sperm plasma membrane, so effective solubilization is critical. Use RIPA buffer or a detergent cocktail containing 1 percent Triton X-100 plus 0.1 percent SDS with protease inhibitors; avoid mild lysis conditions that leave membrane proteins in the pellet. Sonication or freeze-thaw cycles after detergent extraction improve yield. For whole sperm, wash cells thoroughly to remove seminal plasma proteins before lysis. Load 20-30 µg total protein per lane. If signal is weak, enrich membrane fractions by ultracentrifugation before solubilization rather than increasing total lysate load, which raises background.

Does ADAM-20 have splice isoforms I should know about?

ADAM-20 does not have well-characterized splice isoforms reported in UniProt or major databases. The canonical 726-residue sequence appears to be the predominant transcript in human testis. However, proteolytic processing is physiologically relevant: the prodomain is cleaved during sperm maturation, and further ectodomain shedding may occur. You may observe multiple bands on Western blot representing precursor, mature, and shed forms rather than true splice variants. If you detect unexpected band patterns, consider that ADAM family proteins often undergo complex post-translational modification including glycosylation and furin-mediated cleavage, particularly in the testicular environment.

Can I use this antibody for immunofluorescence on human sperm cells?

Immunofluorescence is a validated application across the Triple Point Biologics antibody range, and ADAM-20's plasma membrane localization on spermatozoa makes it a suitable IF target. Fix sperm in 4 percent paraformaldehyde, permeabilize with 0.1-0.2 percent Triton X-100, and start with a 1:100-1:200 dilution of this antibody. ADAM-20 should localize to the sperm head and midpiece plasma membrane. Include non-permeabilized controls to assess surface versus internal staining. Because this is a polyclonal antibody, pre-adsorption with excess blocking peptide serves as a useful specificity control alongside secondary-only and isotype controls.

What positive control tissue should I use for ADAM-20 Western blots?

Human testis lysate is the optimal positive control, as ADAM-20 expression is highly restricted to testicular germ cells and spermatozoa. Commercial human testis lysates are available from several vendors if primary tissue is inaccessible. Isolated human sperm preparations also work but may give weaker signals depending on purity and maturation status. Avoid using ubiquitous tissues like HeLa, HEK293, or liver as controls; ADAM-20 is not expressed there. If validating cross-reactivity with rodent samples, mouse or rat testis is the appropriate positive control. Include molecular weight markers spanning 70-100 kDa for accurate band identification.

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. The 100 µg pack size is suitable for dividing into 10-20 µL aliquots depending on your experimental frequency. Once thawed, an aliquot can be held at 4°C for up to one month if sodium azide is present as preservative; check the datasheet for formulation details. Do not store diluted antibody in working buffer for more than a week. Under proper storage at -20°C, rabbit polyclonal antibodies typically retain activity for two to three years from manufacture date.