ADAMTS-13 (Recombinant)

Frequently Asked Questions

What is the expected molecular weight of recombinant ADAMTS-13 on SDS-PAGE or Western blot?

The full-length human ADAMTS-13 polypeptide (1,427 aa) has a predicted molecular weight of ~145 kDa, but the HEK293-expressed recombinant runs at approximately 160–180 kDa under reducing SDS-PAGE conditions due to N-linked glycosylation at multiple sites. This glycosylated form is consistent with the endogenous secreted enzyme. If you are running a non-reducing gel, expect potential disulfide-linked migration artifacts. For Western blot validation using the matched rabbit polyclonal RP-ADAMTS13, the 160–180 kDa band is the expected positive control signal.

What processing or isoform state is this recombinant ADAMTS-13 — pro-form or mature cleaved enzyme?

This recombinant is expressed in HEK293 cells and undergoes pro-domain removal during secretion, yielding the mature, active form characteristic of the endogenously processed enzyme. It is not a pro-enzyme requiring exogenous activation. The furin-like convertase cleavage that removes the pro-domain occurs intracellularly in HEK293 cells, paralleling the native processing pathway. Researchers designing inhibitor screens or substrate cleavage assays should note they are working with the constitutively active mature metalloprotease, not a zymogen, which eliminates the need for an activation step.

What substrate does recombinant ADAMTS-13 cleave and at which bond?

ADAMTS-13 cleaves ultra-large von Willebrand factor (vWF) multimers at the Tyr1605–Met1606 peptide bond within the vWF A2 domain. For in vitro activity assays, the standard minimal substrate is the 73-amino acid peptide FRETS-vWF73, a fluorescence-resonance energy transfer peptide spanning the A2 domain cleavage site. Cleavage of FRETS-vWF73 generates a measurable increase in fluorescence (excitation ~340 nm, emission ~440 nm). Full-length vWF or recombinant vWF A2 domain fragments are also used for more physiological multimer reduction assays under defined shear conditions.

What buffer conditions does recombinant ADAMTS-13 need for maximum activity in a cleavage assay?

The storage buffer is 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 5 mM CaCl₂. For activity assays, ADAMTS-13 is active across pH 7.0–8.0, with optimal activity near pH 7.5. Maintain 5 mM CaCl₂ in the assay buffer; as a zinc-dependent metalloprotease its activity is sensitive to metal chelators — avoid EDTA or EGTA entirely. 1 mM DTT or 1 mM β-mercaptoethanol is tolerated and can improve stability over long incubations. BSA (0.1–0.5%) in the assay buffer reduces nonspecific adsorption to tube walls at low enzyme concentrations.

What starting concentration of recombinant ADAMTS-13 should I use for a FRETS-vWF73 fluorescence activity assay?

For a standard FRETS-vWF73 assay, a useful starting point is 50–200 nM recombinant ADAMTS-13 with 1–2 µM FRETS-vWF73 substrate in 50 mM Tris-HCl pH 7.5, 5 mM CaCl₂, 0.1% BSA at 37°C. Under these conditions, cleavage is typically linear for 30–60 minutes, allowing reliable initial velocity measurements. For IC₅₀ determination of inhibitors, titrate enzyme to the lowest concentration still giving a robust, reproducible signal — typically 25–50 nM — to avoid substrate depletion artifacts. Confirm linearity with respect to enzyme concentration before committing to a full inhibitor panel.

Can I use recombinant ADAMTS-13 as a positive control for Western blot with the RP-ADAMTS13 antibody?

Yes, and this is a validated use case. Load 50–100 ng of this recombinant per lane on a standard SDS-PAGE gel alongside your cell lysate or plasma samples. The matched rabbit polyclonal RP-ADAMTS13 (SKU: RP-ADAMTS13) was raised against an ADAMTS-13 fragment and is guaranteed compatible with this recombinant for Western blot positive control applications. You will see a clean band at approximately 160–180 kDa. This pairing also serves directly for antibody lot validation, ensuring your RP-ADAMTS13 antibody performs before committing it to experimental samples.

How much recombinant ADAMTS-13 should I load for a Western blot positive control lane?

50–100 ng per lane is the recommended loading range when using RP-ADAMTS13 at a 1:1,000–1:2,000 dilution. At 50 ng you should see a strong, unambiguous band at ~160–180 kDa without obscuring nearby molecular weight markers. If you are troubleshooting a new membrane or detection system, run a small titration: 25, 50, 100, and 200 ng in parallel. The recombinant is >90% pure by SDS-PAGE, so the dominant band will be ADAMTS-13 with minimal background, making it straightforward to set exposure times for your experimental lanes.

How should I dilute, aliquot, and store recombinant ADAMTS-13 to preserve activity long-term?

The protein is supplied in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 5 mM CaCl₂ and is stable at −20°C for at least 12 months when stored as single-use aliquots. Repeated freeze-thaw cycles measurably reduce metalloprotease activity — prepare working aliquots at the volume you consume in one experiment and avoid returning thawed material to the freezer. For dilutions below ~10 nM, add carrier protein (0.1–0.5% BSA) to prevent adsorptive losses to tube surfaces. Do not dilute into EDTA-containing buffers; zinc chelation will inactivate the enzyme irreversibly.