RECK (Recombinant)

Frequently Asked Questions

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

RECK has a predicted molecular weight of ~110 kDa based on its 971-amino-acid sequence, but the mature glycoprotein routinely migrates at approximately 130–150 kDa on reducing SDS-PAGE due to extensive N-linked glycosylation. This HEK293-expressed recombinant is produced under conditions that support native glycosylation, so expect the higher apparent MW. If you treat the sample with PNGase F prior to electrophoresis, the band collapses toward the predicted ~110 kDa core, which is a useful confirmation. Purity is >95% by SDS-PAGE.

Is recombinant RECK the full-length protein or a truncated ectodomain fragment?

This product is the soluble ectodomain of human RECK (UniProt O95980), expressed in HEK293 cells without the C-terminal GPI attachment signal. Removing the GPI anchor yields a secreted, soluble form while preserving all functional cysteine-rich and Kazal-type domains relevant to MMP inhibition and Wnt7/ADGRA2 binding. The construct retains the disulfide architecture of the native ectodomain, making it directly applicable to biochemical binding and inhibition experiments without detergent solubilization.

Which MMPs does recombinant RECK inhibit and what substrates work for measuring that activity?

RECK has established inhibitory activity against MMP-2, MMP-9, and MT1-MMP (MMP-14). For activity assays, fluorogenic gelatin-derived substrates such as Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH₂ are commonly used with MMP-2 or MMP-9 as the target proteinase. Set up a standard MMP activity reaction in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10 mM CaCl₂, 0.05% Brij-35, then titrate RECK recombinant from 10–500 nM against a fixed concentration of activated MMP (typically 1–5 nM). IC₅₀ values in the literature for RECK vs. MMP-2 fall in the low-nanomolar to sub-nanomolar range under these conditions.

What starting concentration of recombinant RECK should I use in an MMP inhibition assay?

A practical starting range is 50–200 nM recombinant RECK for initial dose-response experiments against activated MMP-2 or MMP-9 at 2–5 nM. Dilute the stock into assay buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10 mM CaCl₂, 0.05% Brij-35) and pre-incubate RECK with the MMP for 15–30 minutes at 37°C before adding substrate. Avoid introducing the glycerol-containing storage buffer at >1% final volume, as higher glycerol concentrations can depress fluorescence signal. Prepare single-use aliquots before freezing to prevent activity loss from repeated freeze-thaw.

Can I use recombinant RECK as a positive control for Western blot with the matched TPB anti-RECK antibody (RP-RECK)?

Yes — this is the intended use case. Load 20–50 ng of recombinant RECK per lane alongside your cell lysate samples. The protein migrates at ~130–150 kDa under reducing conditions, giving a clean reference band for size confirmation. RP-RECK (/anti-reck-rabbit-polyclonal-antibody) is a rabbit polyclonal raised and validated in the same laboratory that produced this recombinant, so epitope compatibility is confirmed rather than assumed. Recommended antibody dilution for Western blot is 1:500–1:2000; secondary: HRP-conjugated anti-rabbit IgG at 1:5000–1:10000.

How much recombinant RECK should I load for a Western blot positive control with RP-RECK?

20–50 ng per lane is sufficient for a clear, non-saturating band when using RP-RECK at 1:1000 dilution with standard ECL detection. At 50 ng you will see a robust ~130–150 kDa band that serves as both a size marker reference and an antibody specificity control. If you are running a high-sensitivity detection system (e.g., femtogram-range HRP substrates), reduce the load to 5–10 ng to avoid bloom. Because recombinant RECK and the RP-RECK antibody originate from the same TPB workflow, this pairing is also suitable for antibody lot-to-lot validation.

What buffer conditions are optimal for RECK binding studies with Wnt7 or ADGRA2 ectodomain by SPR or BLI?

For SPR or biolayer interferometry, exchange recombinant RECK into HBS-P+ (10 mM HEPES pH 7.4, 150 mM NaCl, 0.05% P-20 surfactant) or PBS + 0.05% Tween-20 via buffer exchange spin column (10 kDa MWCO). The storage buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol) is incompatible with most SPR running buffers at high glycerol content. RECK is stable after a single buffer exchange step when kept at 4°C for up to 48 hours. For Wnt7a/ADGRA2 binding titrations, a RECK surface density of ~500–1000 RU or equivalent BLI loading is a reasonable starting point.

How should I store and handle recombinant RECK to maintain activity over time?

Store at -20°C in single-use aliquots to avoid repeated freeze-thaw cycles, which progressively reduce activity of cysteine-rich proteins. Upon receipt, spin the vial briefly, then aliquot into volumes matching your typical experiment (e.g., 5–10 µg aliquots) before returning to -20°C. For short-term use within 1–2 weeks, the protein is stable at 4°C in its storage buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol). Do not dilute the stock until just before use; carrier protein (0.1% BSA) can be added to diluted working solutions to minimize adsorption to tube walls at concentrations below ~10 nM.