Anti-ADAM-11 Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight should I expect for ADAM-11 on Western blot?

ADAM-11 migrates at approximately 90-100 kDa on Western blot, somewhat higher than its predicted mass of 85 kDa for the 769-amino acid full-length protein. This apparent shift is due to extensive N-glycosylation of the extracellular domain. You may observe additional lower-molecular-weight bands representing proteolytic fragments or incompletely glycosylated forms, particularly in brain lysates where endogenous protease activity can be high. Running samples under reducing conditions and including protease inhibitors in your lysis buffer will help preserve the full-length species.

Is ADAM-11 catalytically active like other ADAM family metalloproteases?

No. ADAM-11 is catalytically inactive due to critical substitutions in the zinc-binding motif of its metalloprotease-like domain. Unlike proteolytically active family members such as ADAM-10 or ADAM-17, ADAM-11 functions exclusively as a cell adhesion molecule and integrin ligand at neuronal synapses. This distinction is important when designing experiments: ADAM-11 will not exhibit metalloprotease activity in substrate cleavage assays, and its biological role centers on protein-protein interactions rather than enzymatic processing. The antibody recognizes the protein regardless of catalytic status.

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

Start at 1:1000 dilution in blocking buffer for Western blot, which is our validated concentration for this antibody. Brain tissue lysates and transfected cell lysates expressing ADAM-11 typically yield clean bands at this dilution with overnight incubation at 4°C. If your signal is weak or your sample has low endogenous ADAM-11 expression, you can increase concentration to 1:500. For non-neuronal tissues where expression may be minimal, load 30-50 µg total protein per lane and consider concentrating to 1:500 as a starting point before further optimization.

Does this antibody work in mouse and rat brain samples?

Cross-reactivity with mouse and rat ADAM-11 is predicted based on sequence homology but not yet validated in our hands. Human ADAM-11 shares approximately 85-90 percent identity with mouse and rat orthologs across the immunogenic region. If working with rodent samples, plan to run a pilot blot with positive control lysate from mouse or rat cerebellum, where ADAM-11 is abundantly expressed at basket cell terminals. Expect the same 90-100 kDa band if cross-reactivity holds. We recommend confirming specificity with a blocking peptide or siRNA knockdown in your model system.

What tissue or cell type should I use as a positive control for ADAM-11?

Human or rodent brain tissue, particularly cerebellum, serves as the strongest positive control due to high endogenous ADAM-11 expression at basket cell presynaptic terminals. Whole-brain lysate from adult tissue works well. For cell culture controls, neuronal cell lines such as SH-SY5Y or differentiated neuroblastoma cells express detectable levels. HEK293 cells transiently transfected with an ADAM-11 expression construct provide a robust overexpression control. Avoid using non-neuronal tissues like liver or kidney as positive controls; ADAM-11 expression outside the central nervous system is minimal and may yield false-negative results.

Can I use this antibody for immunofluorescence on neurons?

Yes, this antibody is suitable for immunofluorescence based on brand-wide validation, though optimal dilution for IF has not been determined specifically for the ADAM-11 clone. Start at 1:200 dilution for cultured neurons or brain sections. Because ADAM-11 localizes to presynaptic membranes, you should see punctate staining at synaptic sites, particularly in cerebellar basket cell terminals. Use a well-characterized synaptic marker like synaptophysin or bassoon for co-localization studies. Antigen retrieval may improve signal in fixed tissue sections; citrate buffer pH 6.0 is a reasonable starting method for membrane proteins.

How should I store the antibody and how long will it remain stable?

Store the antibody at -20°C in single-use aliquots to avoid repeated freeze-thaw cycles, which can reduce titer and increase background. The 100 µg pack size is sufficient for approximately 100 Western blots at 1:1000 dilution; aliquot into 10-20 µL volumes based on your typical usage. Once thawed, an aliquot can be kept at 4°C for up to one month if sodium azide preservative is present, though we recommend storing working aliquots no longer than two weeks. Do not store diluted antibody in blocking buffer for more than 24 hours.

Why am I seeing multiple bands on my ADAM-11 Western blot?

Multiple bands are common with ADAM-11 and typically reflect glycosylation heterogeneity and proteolytic processing. The primary band at 90-100 kDa represents full-length glycosylated ADAM-11. Lower-molecular-weight bands around 70-80 kDa may be incompletely glycosylated precursors or ectodomain-shed fragments generated by endogenous proteases. To minimize artifactual degradation, prepare lysates quickly on ice with a cocktail of protease inhibitors including metalloprotease inhibitors like EDTA or 1,10-phenanthroline. If bands persist under these conditions, they likely represent biologically relevant processing intermediates rather than sample degradation artifacts.