Anti-ADAM-2 Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight band should I expect for ADAM-2 on a Western blot?

Full-length ADAM-2 has a calculated molecular weight of approximately 82 kDa based on its 735-residue sequence. However, ADAM-2 undergoes post-translational processing and glycosylation, so you may observe the mature form migrating between 90-110 kDa on reducing SDS-PAGE. In testis and sperm lysates, you might also detect processing intermediates or cleavage products at lower molecular weights. Running a positive control lysate from human testis tissue alongside your samples will help confirm the expected band pattern for your specific gel conditions.

What is the recommended starting dilution for ADAM-2 antibody in Western blot?

Start at 1:1000 dilution in blocking buffer for Western blot applications, which has been validated for this antibody. Depending on your sample type and protein loading amount, you may need to optimize between 1:500 and 1:2000. Testis lysates typically yield strong signal at 1:1000, while sperm preparations may require higher antibody concentrations due to lower total protein input. Incubate overnight at 4°C for optimal signal. Always include a no-primary control to assess non-specific binding of your secondary antibody.

Which human tissues or cell lines are good positive controls for ADAM-2?

Human testis tissue lysate is the most reliable positive control, as ADAM-2 expression is predominantly restricted to testis and mature spermatozoa. If testis tissue is unavailable, purified human sperm preparations can serve as an alternative, though protein yield per sample may be lower. ADAM-2 is classified as a cancer/testis antigen, so certain melanoma, lung, or bladder cancer cell lines may show ectopic expression, but this varies widely by cell line and passage. Negative controls should include somatic tissues such as liver, brain, or skeletal muscle, where ADAM-2 is not normally expressed.

Is this ADAM-2 antibody likely to cross-react with mouse or rat samples?

This antibody has been validated only in human samples. Cross-reactivity with rat is listed as potential based on sequence homology, but has not been experimentally verified. Mouse ADAM-2 shares significant conservation in the metalloprotease-like and disintegrin domains, but epitope accessibility may differ. If you plan to use rat, mouse, or other rodent samples, we recommend testing a dilution series starting at 1:500 with appropriate testis lysate positive controls. Be prepared to optimize blocking conditions and consider running human testis lysate in parallel to confirm antibody performance.

Can I use this antibody for immunofluorescence on sperm cells?

This rabbit polyclonal has been validated for Western blot; immunofluorescence applications on sperm are feasible but will require optimization. ADAM-2 localizes to the sperm plasma membrane, particularly in the head region. For intact sperm, permeabilization with 0.1-0.2% Triton X-100 may be necessary depending on epitope accessibility. Start with a 1:100 to 1:200 dilution and include a no-primary control. Methanol or paraformaldehyde fixation can both work, but methanol often preserves sperm morphology better. Surface staining without permeabilization may be attempted first to assess membrane-accessible epitopes.

Does ADAM-2 have multiple isoforms I should be aware of?

The primary UniProt entry (Q99965) describes a single 735-amino acid isoform, but ADAM-2 undergoes extensive proteolytic maturation. The protein is synthesized as a precursor with a prodomain that is cleaved during sperm maturation in the epididymis. Additionally, ADAM-2 exists as part of a heterodimeric complex with ADAM-3 (fertilin alpha) on the sperm surface, so co-immunoprecipitation studies may pull down both subunits. Alternative splicing has not been extensively reported for human ADAM-2. Focus on the mature processed form when interpreting blots from ejaculated sperm versus testicular tissue.

What sample preparation considerations are important for ADAM-2 detection?

ADAM-2 is a membrane-anchored metalloprotease, so complete solubilization is critical. Use RIPA buffer or a detergent-containing lysis buffer with at least 1% NP-40 or Triton X-100. For sperm samples, wash cells thoroughly to remove seminal plasma proteins before lysis. Include protease inhibitors to prevent post-lysis degradation, particularly metalloprotease inhibitors such as EDTA or 1,10-phenanthroline if you want to preserve full-length protein. Sonication or mechanical disruption improves yield from sperm, which have tightly compacted membranes. Load 20-40 µg total protein per lane for Western blot.

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 sufficient for approximately 100 Western blots at 1:1000 dilution. Once thawed, an aliquot can be kept at 4°C for up to one month if sodium azide is present as a preservative. For long-term storage beyond the initial shelf life, keep aliquots at -80°C. Do not store diluted antibody in blocking buffer for more than one week. Check the product datasheet for the specific formulation and any additional stabilizers included.