ADAM-23 (Recombinant)

Frequently Asked Questions

What molecular weight does recombinant ADAM-23 run at on SDS-PAGE or Western blot?

The ADAM-23 coding sequence predicts a theoretical molecular weight of approximately 93 kDa from its 832 amino acids, but the HEK293-expressed recombinant typically migrates at 100–115 kDa under reducing SDS-PAGE conditions due to N-linked glycosylation across multiple extracellular domain sites. Under non-reducing conditions, intramolecular disulfide bonds in the cysteine-rich domain can compress apparent mobility slightly. When running this protein as a positive control alongside RP-ADAM23 on Western blot, expect the primary band in the 100–115 kDa window; faint lower bands may reflect partial deglycosylation during sample prep.

Is recombinant ADAM-23 a full-length protein or a processed extracellular domain fragment?

The REC-ADAM23 construct covers the mature ectodomain — prodomain through cysteine-rich region — and excludes the transmembrane anchor and cytoplasmic tail. This design reflects the fact that ADAM-23's functionally characterised interactions (with LGI1 and ADAM22 in the trans-synaptic complex) are mediated entirely by extracellular domains. Full-length ADAM-23 undergoes prodomain removal during biosynthetic trafficking; the recombinant is supplied post-prodomain cleavage, representing the processed, membrane-competent form relevant to binding and scaffolding experiments.

Does ADAM-23 have metalloprotease activity, and what substrates should I use to test it?

ADAM-23 is a catalytically inactive ADAM family member. Its metalloprotease domain lacks the consensus HExxHxxGxxH zinc-binding motif required for catalysis, so it will not cleave generic ADAM substrates such as Mca-PLAQAV-Dpa-RSSSR-NH2 or quenched fluorescent peptides based on TNF-α or EGF-R sequences. Do not use standard ADAM activity assays to evaluate this protein — they will return a flat signal, which is the expected result. ADAM-23 is best characterised in binding assays (SPR, co-IP, pull-down) probing its disintegrin-like or cysteine-rich domain interactions with LGI1 or ADAM22.

What buffer conditions are recommended for ADAM-23 binding assays or pull-down experiments?

The protein is supplied in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol. For pull-down or co-immunoprecipitation experiments probing LGI1 or ADAM22 binding, this buffer is compatible as-is; glycerol stabilises the cysteine-rich domain during incubation. If you are diluting into a cell lysate binding buffer (e.g., 25 mM HEPES pH 7.4, 150 mM NaCl, 0.5% NP-40), keep final glycerol below 2% to avoid viscosity artefacts. Because ADAM-23 is not a metalloproteinase, CaCl2 supplementation is not required but is not harmful at 1–2 mM.

What starting concentration of recombinant ADAM-23 should I use for a protein–protein interaction binding assay?

For surface plasmon resonance or bio-layer interferometry with LGI1 or ADAM22 as the analyte, a concentration series of 5–500 nM ADAM-23 in running buffer (PBS pH 7.4, 0.05% Tween-20) gives a workable signal window based on published KD values in the low-nanomolar range. For pull-down experiments from HEK293 or neuronal lysates, 0.5–2 µg of recombinant protein per 500 µg total lysate is a reasonable starting point. Titrate downward if non-specific binding to beads is observed; the >95% purity specification minimises but does not eliminate background.

Can I use REC-ADAM23 as a positive control for Western blot when validating the RP-ADAM23 rabbit polyclonal antibody?

Yes — this is one of the primary intended uses. Load 20–50 ng of REC-ADAM23 per lane alongside your cell lysate samples. The matched antibody RP-ADAM23 (see /anti-adam-23-rabbit-polyclonal-antibody) is raised against the same ectodomain region present in this recombinant, so epitope accessibility is guaranteed. Run under reducing conditions; expect a clean band at 100–115 kDa. This pairing is also useful for antibody titration: a dilution series of RP-ADAM23 (typically 1:500–1:5000) against 30–50 ng recombinant defines the linear detection range before applying the antibody to tissue lysates.

How much REC-ADAM23 should I load for a Western blot positive control lane?

20–50 ng per lane is sufficient for a strong, clean signal with RP-ADAM23 at 1:1000–1:2000 dilution on a standard PVDF membrane. At 50 ng you will see a robust band at approximately 100–115 kDa with minimal background. Avoid loading above 100 ng unless you are specifically characterising antibody saturation — overloading a glycosylated recombinant can produce a smeared upper band that may be mistaken for a doublet. Denature samples at 95°C for 5 minutes in reducing Laemmli buffer; the disulfide-rich cysteine domain requires complete reduction for sharp migration.

How should I store and handle REC-ADAM23 to maintain activity across multiple experiments?

REC-ADAM23 is supplied in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol and shipped on dry ice. Upon receipt, aliquot immediately into single-use volumes (typically 5–10 µg per tube) and store at −20°C. Avoid repeated freeze-thaw cycles; each cycle can promote aggregation of the cysteine-rich domain and reduce binding competence measurable in pull-down assays. For short-term use (up to 48 hours), the protein is stable at 4°C. Do not dilute working aliquots more than 1:10 below the supplied concentration without adding 0.1% BSA as a carrier to prevent adsorptive loss to tube surfaces.