ADAM-22 (Recombinant)

Frequently Asked Questions

What is the expected molecular weight of recombinant ADAM-22 on SDS-PAGE and Western blot?

The full-length human ADAM-22 protein (906 aa) has a predicted unmodified MW of ~102 kDa. Because this recombinant construct is the extracellular fragment produced in HEK293 cells — spanning the prodomain through the EGF-like domain — and carries mammalian N-linked glycosylation, expect a diffuse band running between 110–130 kDa under reducing conditions on SDS-PAGE. Glycosylation heterogeneity is the primary source of smearing. Treating the sample with PNGase F prior to electrophoresis will sharpen the band and shift it closer to the predicted polypeptide backbone mass.

Does recombinant ADAM-22 have metalloproteinase activity and what is its catalytic status?

ADAM-22 is catalytically inactive. The metalloproteinase-like domain lacks the canonical HExxHxxGxxH zinc-binding consensus required for proteolysis — a glutamic acid critical for catalysis is absent. Do not use this recombinant in substrate-cleavage assays expecting protease activity. Its functional relevance lies in protein–protein interactions: it binds LGI1 with high affinity and can engage integrins through its disintegrin-like domain. Assay design should therefore focus on binding, co-precipitation, or surface plasmon resonance formats rather than fluorogenic peptide cleavage.

What assay formats are appropriate for studying ADAM-22 and LGI1 binding with this recombinant?

Solid-phase binding assays (ELISA-style capture), co-immunoprecipitation, and surface plasmon resonance (SPR) are all well-suited. For a direct binding ELISA, coat recombinant ADAM-22 at 1–4 µg/mL in PBS overnight at 4°C, then titrate recombinant LGI1 as the analyte. For SPR, immobilise ADAM-22 via amine coupling and flow LGI1 at concentrations spanning the reported Kd (~1–10 nM range). The storage buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol) is compatible with both formats after dilution into assay buffer; glycerol carry-over above 0.05% can affect SPR baselines, so dilute accordingly.

What buffer conditions should I use when working with recombinant ADAM-22 in binding experiments?

The supplied storage buffer is 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol. Note that ADAM-22 is not a metalloproteinase, so no CaCl2 supplement is included. For binding assays, dilute into a physiologically relevant buffer such as PBS + 0.05% Tween-20 or HEPES-buffered saline (25 mM HEPES pH 7.4, 150 mM NaCl, 0.005% surfactant P20 for SPR). Avoid reducing agents above 1 mM DTT, as the disintegrin and cysteine-rich domains contain intramolecular disulfide bonds critical for maintaining the LGI1-binding competent conformation.

What starting concentration of recombinant ADAM-22 should I use for a protein–protein interaction pull-down experiment?

For bead-based pull-down, couple 5–10 µg of recombinant ADAM-22 per reaction to Ni-NTA or streptavidin resin (depending on your tag format) and use 500 µg–1 mg total cell lysate as input. If you are titrating LGI1 analyte in a direct binding assay, begin at 200 nM and perform a 2-fold serial dilution across 8–10 concentrations to establish a binding curve. Non-specific binding controls using a structurally unrelated recombinant at matched concentrations are advisable, given the adhesion-molecule nature of ADAM-22 and its potential for off-target integrin interactions.

Can I use recombinant ADAM-22 as a positive control for Western blot with the matched RP-ADAM22 antibody?

Yes — this is one of the primary validated use cases. Load 25–50 ng of recombinant ADAM-22 per lane alongside your cell lysate samples. The matched rabbit polyclonal antibody RP-ADAM22 (/anti-adam-22-rabbit-polyclonal-antibody) is raised against the extracellular region covered by this recombinant construct, so epitope overlap is guaranteed. Expect a band at 110–130 kDa (glycosylated form) in the recombinant lane. This band serves as an unambiguous size marker for endogenous ADAM-22 in neuronal lysates, where the protein runs at a similar apparent MW.

How much recombinant ADAM-22 should I load to optimise the RP-ADAM22 antibody titration on Western blot?

Start with a 3-point titration: 10 ng, 25 ng, and 100 ng per lane. At 25 ng, RP-ADAM22 typically produces a clean signal with primary antibody dilutions in the 1:1,000–1:2,000 range (HRP-conjugated secondary, ECL detection). Overloading above 200 ng can cause smearing due to glycosylation heterogeneity and may obscure the endogenous band in adjacent lysate lanes. For antibody lot-to-lot validation, the 25 ng lane paired with a matched neuronal cell lysate positive control provides a reliable two-band reference pattern.

How should I store and handle recombinant ADAM-22 to maintain activity and prevent degradation?

Store at -20°C in single-use aliquots immediately upon receipt. The supplied 10% glycerol cryoprotectant stabilises the protein through freeze-thaw, but repeated cycling will cause aggregation and loss of binding-competent conformation — aliquot at first thaw if the stock vial is not pre-divided. On ice, the protein is stable for up to 8 hours during working sessions. Avoid diluting to below ~10 µg/mL in the absence of a carrier protein (0.1% BSA can be added); at very low concentrations, adsorptive loss to tube surfaces becomes significant. Purity is >90% by SDS-PAGE and endotoxin is <0.1 EU/µg by LAL.