Anti-ADAM-30 Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight band should I expect for ADAM-30 on Western blot?

ADAM-30 has a predicted molecular weight of approximately 87 kDa based on the 790-amino acid sequence. However, ADAM family proteins often migrate at apparent masses that differ from predicted values due to glycosylation and proteolytic processing. You may observe bands between 80-100 kDa depending on post-translational modifications. Because ADAM-30 localizes to late endosomes and may undergo maturation cleavage, lysates from different tissue sources or cell lines could yield additional lower-mass bands representing processed forms. Include a positive control lysate from cells known to express ADAM-30, and consider treating samples with deglycosylases if band interpretation is ambiguous.

What dilution should I start with for ADAM-30 Western blot?

We recommend starting at 1:1000 dilution for Western blot applications with this rabbit polyclonal antibody. This concentration has been validated in our hands and provides a good balance between signal and background for most mammalian cell lysates. If your ADAM-30 expression level is low or you are working with tissue lysates rather than overexpression models, you may need to titre up to 1:500. Conversely, if you encounter high background, dilute to 1:2000. Because ADAM-30 is an endosomal protein, enriching for membrane fractions rather than using whole-cell lysates may improve signal-to-noise ratio.

Will this ADAM-30 antibody cross-react with mouse or rat samples?

This antibody has been validated against human ADAM-30. Cross-reactivity with mouse and rat is predicted based on sequence homology in the immunogen region, but has not been experimentally validated by Triple Point Biologics. Human and mouse ADAM-30 share approximately 75-80 percent sequence identity, suggesting potential cross-reactivity. However, if you are working with rodent samples, we recommend treating the first experiment as a validation run. Include a human-derived positive control in parallel, and verify that the observed band corresponds to the expected molecular weight for rodent ADAM-30, which may differ slightly from the human ortholog.

Does ADAM-30 have multiple isoforms I need to account for?

ADAM-30 is primarily represented by a single full-length isoform in UniProt (Q9UKF2), spanning 790 amino acids. However, like other ADAM family members, ADAM-30 undergoes proteolytic maturation that removes the prodomain, generating a catalytically active form. Depending on your cell type and lysis conditions, you may detect both the zymogen and mature protease on Western blot. Additionally, because ADAM-30 functions in the endolysosomal compartment where proteolysis is active, you may observe degradation products or C-terminal fragments. If studying ADAM-30 processing is central to your work, consider using protease inhibitor cocktails during lysis and comparing reducing versus non-reducing conditions.

Can I use this antibody for immunofluorescence to visualize ADAM-30 in endosomes?

Triple Point Biologics antibodies are routinely validated for immunofluorescence applications, and this polyclonal format is generally compatible with IF protocols. Because ADAM-30 localizes to late endosomes, you should see punctate perinuclear staining in cells expressing the protein. Use methanol or paraformaldehyde fixation followed by permeabilization with 0.1-0.2 percent Triton X-100. Start with a 1:100 to 1:200 dilution and co-stain with late endosome markers such as Rab7 or LAMP1 to confirm colocalization. Keep in mind that ADAM-30 expression is often low in non-neuronal cells, so select a cell model with documented ADAM-30 expression or consider transient overexpression for method development.

What positive control should I use to validate ADAM-30 detection?

ADAM-30 expression is relatively restricted compared to ubiquitous ADAMs like ADAM10 or ADAM17. Brain tissue lysates, particularly from cortex or hippocampus, are reasonable positive controls given ADAM-30's role in amyloid precursor protein processing and relevance to neurodegenerative pathways. Alternatively, HEK293 or SH-SY5Y neuroblastoma cells express detectable endogenous ADAM-30. If endogenous levels are too low for reliable detection in your hands, transiently transfect cells with an ADAM-30 expression construct as a positive control. A lysate from non-transfected cells or ADAM-30 knockdown cells serves as a useful negative control to assess antibody specificity.

How should I prepare lysates to preserve ADAM-30 for Western blot?

ADAM-30 is a membrane-associated metalloprotease localized to endosomes, so lysis buffer composition matters. Use RIPA or a similar detergent-based buffer containing 1 percent NP-40 or Triton X-100 to solubilize membrane proteins effectively. Include a broad-spectrum protease inhibitor cocktail, and consider adding a metalloprotease inhibitor such as 10 mM EDTA or 1 mM 1,10-phenanthroline to prevent autocatalytic degradation or cleavage by other metalloproteases. Because ADAM-30 functions within the acidic endolysosomal compartment, avoid freeze-thaw cycles that may compromise membrane integrity. Prepare fresh lysates when possible, or snap-freeze aliquots in liquid nitrogen and store at -80°C for long-term use.

How should I store this ADAM-30 antibody and how long is it stable?

Store the antibody at -20°C upon arrival. Rabbit polyclonal antibodies in standard formulation with carrier protein and preservative are stable for at least 12 months under these conditions. Avoid repeated freeze-thaw cycles, which can reduce titre and increase aggregation. For routine use, aliquot the stock into single-use volumes and keep a working aliquot at 4°C for up to one month. Do not store diluted antibody; prepare fresh working dilutions in blocking buffer for each experiment. If you observe decreased signal or increased background over time, this typically indicates antibody degradation, and a fresh aliquot should be thawed.