Anti-ADAM-1 Rabbit Polyclonal Antibody

Frequently Asked Questions

What is the expected molecular weight for ADAM-1 on Western blot?

Full-length ADAM-1a runs at approximately 87-90 kDa on reducing SDS-PAGE, corresponding to the 791-amino acid unprocessed form. However, ADAM-1 undergoes proteolytic processing during sperm maturation, yielding fragments that may appear between 50-70 kDa depending on the cleavage state. In testicular lysates you may detect higher molecular weight precursors, while mature sperm extracts typically show processed forms. Post-translational modifications including glycosylation can shift the apparent size by 5-10 kDa above the predicted mass. Always include a positive control from testis or mature sperm to establish the processing pattern in your biological system.

What dilution should I start with for ADAM-1 Western blots?

We recommend starting at 1:1000 dilution for Western blot with overnight incubation at 4°C. This dilution has been validated in our hands using 20-30 µg of testis or sperm lysate per lane. If signal is weak, increase antibody concentration to 1:500 or extend incubation time before concluding the target is absent. Conversely, if you observe high background, dilute to 1:2000. The 100 µg pack size provides sufficient material for approximately 100 blots at the 1:1000 working dilution, assuming 10 mL blocking volume per membrane.

Is this ADAM-1 antibody validated for mouse and rat samples?

This antibody is validated for human ADAM-1a with high confidence. Mouse and rat cross-reactivity are predicted based on sequence homology in the immunogen region but have not been experimentally confirmed in-house. Mouse ADAM-1a (UniProt Q60813) shares approximately 75-80% identity with human orthologues in conserved epitope regions. If working with rodent samples, treat this as an exploratory reagent and include appropriate species-matched positive controls—testis lysate is ideal. We recommend running human and mouse samples in parallel on your first blot to assess cross-reactivity directly in your system.

Can I use this antibody for immunofluorescence on sperm cells?

This rabbit polyclonal is suitable for immunofluorescence applications based on our brand-wide validation protocols, though we have not specifically tested it on sperm. For intact spermatozoa, permeabilization is critical since ADAM-1 contains cytoplasmic and transmembrane domains. Use 0.1-0.5% Triton X-100 for 10-15 minutes after fixation with 4% paraformaldehyde. Start with a 1:100 to 1:200 dilution and expect localization to the posterior head and equatorial segment in mature sperm, consistent with published ADAM-1/fertilin alpha distribution. Include non-permeabilized cells as a negative control to confirm you are detecting intracellular epitopes.

What sample preparation is recommended for detecting ADAM-1 in testis lysates?

Prepare testis lysates in RIPA buffer containing protease inhibitors (EDTA-free if preserving metalloproteinase activity is required, though detection does not require active enzyme). Homogenize thoroughly since ADAM-1 is a transmembrane protein; incomplete solubilization yields weak signal. Include 1% NP-40 or Triton X-100 minimum to extract membrane-associated protein. Load 20-30 µg total protein per lane for initial experiments. Avoid boiling samples excessively—5 minutes at 95°C in reducing sample buffer is sufficient. Testis expresses both precursor and processed forms, so expect multiple bands. Epididymal sperm will show predominantly the mature, processed heterodimer.

Does ADAM-1 form a complex with ADAM-2, and will this antibody detect it?

ADAM-1 functions as a non-covalent heterodimer with ADAM-2 (fertilin beta) in mature sperm, and the complex is required for proper localization and function. This antibody targets ADAM-1a specifically and will not cross-react with ADAM-2. Under reducing Western blot conditions, the heterodimer dissociates and you will detect only ADAM-1 subunits. For co-immunoprecipitation experiments aimed at pulling down the ADAM-1/ADAM-2 complex, use native lysis conditions without SDS and avoid reducing agents. The antibody should work for IP, though we have not formally validated immunoprecipitation applications in our facility.

What positive and negative controls should I use for ADAM-1 experiments?

Positive controls: adult testis lysate or purified mature spermatozoa from the species you are studying. Testis shows robust ADAM-1 expression during spermatogenesis. Negative controls: somatic tissues such as liver, brain, or kidney lysates, where ADAM-1 is not expressed. Additionally, run a no-primary-antibody control to assess non-specific secondary binding. If investigating fertilization biology, consider comparing caput versus cauda epididymal sperm, since ADAM-1 processing occurs during epididymal maturation. Pre-incubating the antibody with immunogen peptide (if available) provides an additional specificity control by eliminating specific signal.

How should I store this antibody and what is the shelf life?

Store the antibody at -20°C in single-use aliquots to avoid freeze-thaw cycles, which can reduce titre and increase aggregation. The lyophilized or glycerol-stabilized format remains stable for at least 12 months at -20°C. Once reconstituted or thawed, aliquots are stable for one month at 4°C with 0.02% sodium azide as preservative, though we recommend using working aliquots within two weeks for optimal performance. Do not store diluted antibody in blocking buffer for more than 24 hours. If you observe precipitate after thawing, centrifuge at 10,000 x g for 30 seconds before use to remove aggregates that cause high background.