ADAM-29 (Recombinant)

Frequently Asked Questions

What molecular weight band should I expect for recombinant ADAM-29 on SDS-PAGE or Western blot?

The ADAM-29 polypeptide backbone is 820 amino acids, giving a predicted unmodified MW of ~92 kDa. Because this recombinant is produced in HEK293 cells, native-like N-linked glycosylation is present across the metalloprotease, disintegrin, and cysteine-rich domains, and the apparent MW on SDS-PAGE typically runs at 100–115 kDa under reducing conditions. Batch-specific observed MW is reported on the Certificate of Analysis. If your band migrates in that range under reducing conditions, the preparation is behaving as expected. Non-reducing conditions may shift migration due to intact disulfide bonds.

Is ADAM-29 catalytically active, and what proteolytic substrates can I use to measure its activity?

ADAM-29 is classified as a metalloprotease-like, enzymatically inactive member of the ADAM family. The canonical HExxHxxGxxH zinc-binding motif is structurally present, but the critical catalytic residues required for proteolysis are absent. This means standard fluorogenic peptide substrates used for active ADAMs (e.g., Mca-PLAQAV-Dpa-NH2 for ADAM-10/17) are not appropriate here. Experimental applications should focus on protein–protein interaction studies, binding assays, or receptor engagement experiments rather than protease activity assays. Confirm your experimental design does not depend on cleavage of a defined substrate.

What buffer conditions are recommended for ADAM-29 recombinant protein in binding or pull-down assays?

The supplied storage buffer is 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, with 5 mM CaCl₂ included to stabilize the metalloprotease-domain fold, consistent with the ADAM family formulation. For pull-down or co-IP experiments, diluting into a physiological binding buffer (25 mM HEPES pH 7.4, 150 mM NaCl, 1 mM CaCl₂, 0.05% Tween-20) is a reasonable starting point. Keep CaCl₂ in your assay buffer at 0.5–1 mM to maintain structural integrity of the metalloprotease scaffold. Avoid EDTA or EGTA, which chelate Ca²⁺ and may destabilize the domain.

What starting concentration should I use for recombinant ADAM-29 in a protein interaction or ELISA capture experiment?

For direct ELISA capture or biolayer interferometry (BLI) binding experiments, a useful starting concentration range is 0.5–5 µg/mL as the immobilized or analyte protein. For co-immunoprecipitation or pull-down, 1–2 µg of recombinant ADAM-29 per reaction is a practical entry point, with the interaction partner at a 2–5 molar excess. Since ADAM-29 is enzymatically inactive, there is no activity-based titration to anchor these concentrations; dose-response optimization against your specific binding partner is recommended. Purity is >90% by SDS-PAGE, so the stated input mass is a reliable approximation of active protein.

What is the expression and processing form of recombinant ADAM-29 — is the prodomain removed?

The recombinant ADAM-29 expressed in HEK293 cells retains the prodomain unless otherwise specified in the lot-specific Certificate of Analysis. In endogenous ADAM biology, the prodomain functions as a cysteine-switch inhibitory segment and is cleaved during or after trafficking through the secretory pathway. Depending on the construct boundaries and whether furin-site processing occurs in HEK293 cells, you may observe either the full-length proform or a processed mature form. Review the CoA for the exact construct boundaries and observed band pattern. This distinction matters if your assay targets a prodomain epitope.

Can I use recombinant ADAM-29 (REC-ADAM29) as a positive control for Western blot with the matched anti-ADAM-29 antibody (RP-ADAM29)?

Yes — REC-ADAM29 and RP-ADAM29 are produced and validated in the same lab specifically for this use case. Loading 50–100 ng of REC-ADAM29 per lane is sufficient to generate a clean positive-control band at ~100–115 kDa under reducing SDS-PAGE conditions with RP-ADAM29 at its recommended dilution (see the antibody datasheet at /anti-adam-29-rabbit-polyclonal-antibody). This pairing is also appropriate for antibody lot-to-lot validation: the recombinant provides a defined, reproducible standard against which antibody sensitivity can be benchmarked across experiments.

How much recombinant ADAM-29 should I load as a Western blot positive control, and at what antibody dilution?

Load 50–100 ng of REC-ADAM29 in a dedicated lane alongside your cell or tissue lysate. At this input, RP-ADAM29 (rabbit polyclonal, SKU RP-ADAM29) at 1:500–1:2000 dilution in 5% non-fat milk/TBST typically yields a well-resolved band at ~100–115 kDa with low background. If you are running a high-percentage gel (10–12%), expect the glycosylated band to compress toward the upper range. Start at 1:1000 and adjust based on signal intensity. The recombinant's >90% purity means the dominant band in your positive-control lane should be ADAM-29 with minimal contaminating species.

How should I handle, dilute, and store recombinant ADAM-29 to preserve stability and avoid activity loss?

REC-ADAM29 is supplied in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 5 mM CaCl₂, and should be stored at −20°C in single-use aliquots. Repeated freeze-thaw cycles degrade the disulfide-bonded domain architecture common to ADAM family proteins and should be strictly avoided. For working dilutions, use the supplied buffer or a compatible assay buffer maintaining CaCl₂ (≥0.5 mM). Prepare dilutions fresh in low-binding tubes; carrier protein (0.1% BSA) can be added for dilutions below 10 µg/mL to minimize surface adsorption losses. Stable for up to 12 months at −20°C from the date of receipt when handled correctly.