TIMP-3 (Recombinant)

Frequently Asked Questions

What molecular weight should I expect for recombinant TIMP-3 on SDS-PAGE or Western blot?

The full-length human TIMP-3 precursor is 211 amino acids with a predicted unglycosylated MW of ~24 kDa. Because this recombinant is produced in HEK293 cells, N-linked glycosylation shifts the apparent MW on reducing SDS-PAGE to approximately 27–30 kDa, consistent with the native secreted form. Under non-reducing conditions, intramolecular disulfide bonds can further alter migration. If you are running a Western blot positive control alongside our matched antibody RP-TIMP3, expect the primary band in the 27–30 kDa range; a faint higher-MW band from glycoform heterogeneity is occasionally observed.

Is this recombinant TIMP-3 the full-length precursor or a processed mature form?

This product corresponds to the full-length human TIMP-3 precursor (UniProt P35625, residues 1–211), including the native signal peptide region. The secreted mature form — generated after signal peptide cleavage — begins at approximately residue 24, so the predominant species in solution represents the processed, secretion-competent protein. No pro-domain truncation is introduced. The HEK293 expression system supports the complete 12-cysteine disulfide framework critical for TIMP-3 folding and its characteristic tight ECM association, which is relevant if you are using this reagent in matrix-binding or surface plasmon resonance assays.

Which MMPs and ADAMs does recombinant TIMP-3 inhibit, and how broad is its selectivity?

TIMP-3 has one of the broadest inhibitory profiles in the TIMP family. Characterised MMP targets include MMP-1, MMP-2, MMP-3, MMP-7, MMP-9, MMP-13, MMP-14 (MT1-MMP), and MMP-15 (MT2-MMP). Beyond classical MMPs, TIMP-3 is the only TIMP family member known to potently inhibit ADAM-family sheddases, including ADAM10 and ADAM17 (TACE). This dual MMP/ADAM inhibitory scope makes it useful for dissecting shedding events from matrix remodeling in the same experiment. If you need to profile selectivity across a panel of zinc-dependent proteases, TIMP-3 covers both sub-families in a single reagent.

What fluorogenic substrate and assay buffer should I use to measure TIMP-3 inhibitory activity?

For MMP inhibition assays, Mca-KPLGL-Dpa-AR-NH2 (a quenched fluorogenic peptide, ex/em ~320/405 nm) is a well-validated substrate for MMP-2, MMP-9, and MMP-13 titration experiments. Run reactions in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10 mM CaCl2, 0.05% Brij-35 at 37°C — the Brij-35 prevents surface adsorption of both the enzyme and inhibitor. Note that TIMP-3's tight ECM-binding character can cause non-specific adsorption to polystyrene microplate surfaces; use BSA-blocked low-binding plates and keep incubation times consistent across replicates to minimise this variable.

What starting concentration and pre-incubation time should I use for a TIMP-3 IC50 assay against MMP-9?

For a standard IC50 titration against MMP-9, start with a TIMP-3 concentration range of 0.1–100 nM (serial 3-fold dilutions, 8–10 points). Pre-incubate TIMP-3 with MMP-9 for 30–60 minutes at 37°C before adding substrate — TIMP-3 is a tight-binding, slow-on inhibitor, and shorter pre-incubation underestimates potency. Reported Ki values for TIMP-3/MMP-9 are in the low-nanomolar to sub-nanomolar range; if your calculated IC50 is significantly higher than 5–10 nM, verify that MMP-9 is fully activated (pro-domain removed) and that the assay buffer contains at least 5 mM CaCl2 for metalloproteinase stability.

What is the storage buffer for recombinant TIMP-3 and can I dilute it directly into cell culture media?

This product is supplied in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM CaCl2, 10% glycerol. For cell-based assays, dilute at least 1:10 into serum-free or low-serum media immediately before use to reduce glycerol to ≤1% (higher concentrations can affect cell viability). Pre-dilute in PBS or assay buffer as an intermediate step if the working concentration requires a smaller final dilution. Protein should be aliquoted upon first receipt, stored at -20°C, and used within 12 months; repeated freeze-thaw cycles measurably reduce inhibitory activity given TIMP-3's disulfide-dependent fold.

Can I use recombinant TIMP-3 as a positive control for Western blot with the RP-TIMP3 antibody?

Yes — this recombinant (REC-TIMP3) was specifically produced to pair with the RP-TIMP3 rabbit polyclonal antibody as a confirmed positive control. Load 20–50 ng of REC-TIMP3 per lane on a standard 12% SDS-PAGE gel under reducing conditions; the antibody detects the glycosylated band at 27–30 kDa with high signal-to-noise at dilutions of 1:1,000–1:2,000. This pairing is also useful for antibody titration experiments — running a dilution series of REC-TIMP3 alongside your cell lysate allows you to bracket the endogenous band and confirm linearity before committing to a full experimental Western blot.

How much recombinant TIMP-3 should I load as a positive control alongside RP-TIMP3 antibody in a Western blot?

For most Western blot setups using RP-TIMP3, 20–50 ng of REC-TIMP3 per lane is sufficient to produce a clean, unambiguous band at 27–30 kDa without overloading. If you are running high-sensitivity chemiluminescent detection (e.g., ECL Plus or a digital imager), start at 20 ng; for colorimetric or less sensitive substrates, 50 ng is a safer starting point. Include this lane adjacent to your cell or tissue lysates so the band position unambiguously identifies endogenous TIMP-3 signal, which can run slightly differently depending on the glycosylation state of the biological sample.