TLL-2 (Recombinant)

Frequently Asked Questions

What molecular weight does recombinant TLL-2 run at on SDS-PAGE and Western blot?

The full-length human TLL-2 sequence (Q9Y6L7) is 1,015 amino acids, giving a predicted unmodified MW of ~114 kDa. Because this recombinant is expressed in HEK293 cells, N-linked glycosylation adds appreciable mass: expect a diffuse band in the range of 120–135 kDa under reducing SDS-PAGE conditions. The smearing is characteristic of heterogeneous glycosylation and is not a purity artefact — >90% purity by SDS-PAGE is confirmed by Coomassie. PNGase F treatment will collapse the band toward the predicted backbone mass if a sharper signal is needed.

Is recombinant TLL-2 the full-length protein or just the catalytic domain, and is the prodomain removed?

This recombinant represents the secreted, processed form of TLL-2. The N-terminal prodomain undergoes autocatalytic or trans removal during HEK293 secretion, consistent with the maturation mechanism described for other BMP-1/Tolloid family members. The active enzyme retains the astacin catalytic domain together with the downstream CUB and EGF-like domains, which are required for substrate recognition and binding. If your experiment requires the zymogen or a defined prodomain-intact construct, this format is not appropriate — contact us to discuss custom expression options.

What substrates does TLL-2 cleave and how do I set up a substrate cleavage activity assay?

TLL-2's best-characterised substrates are the BMP antagonist Chordin and the small leucine-rich proteoglycan Biglycan; cleavage of Chordin at specific sites potentiates BMP signalling. For a direct cleavage assay, incubate recombinant TLL-2 (50–200 nM) with recombinant Chordin (500 nM–1 µM) in assay buffer — 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM CaCl2 — at 37 °C for 1–4 hours, then resolve by SDS-PAGE and detect cleavage fragments. The calcium is essential; chelating agents (EDTA, EGTA) will abolish activity. Include a no-enzyme control and a heat-inactivated TLL-2 control in every experiment.

What buffer should I use for TLL-2 activity assays and what conditions inhibit the enzyme?

The storage buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 5 mM CaCl2) is compatible with direct use in most activity assays; simply dilute into the same buffer base without glycerol for kinetic runs where viscosity is a concern. CaCl2 at 1–5 mM is required for catalytic activity — do not use EDTA- or EGTA-containing buffers. Broad-spectrum metalloprotease inhibitors such as 1,10-phenanthroline (1 mM) or marimastat (10 µM) serve as appropriate positive inhibitor controls. Zinc chelation (e.g., TPEN) at low micromolar concentrations also abolishes activity and can be used to confirm zinc-dependence of any observed cleavage.

What starting enzyme concentration should I use for TLL-2 IC50 inhibitor screening assays?

For IC50 determinations, begin with recombinant TLL-2 at 50–100 nM in assay buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM CaCl2, pH 7.5, 37 °C). This concentration sits within the linear range of activity for substrate cleavage readouts at the 1–4 hour timepoint and provides adequate signal-to-noise without substrate depletion at typical substrate concentrations (500 nM–1 µM Chordin). Pre-incubate enzyme with inhibitor for 30 minutes before substrate addition to allow equilibration. Verify that DMSO vehicle (used for most small-molecule inhibitors) is ≤0.5% v/v, as higher concentrations measurably reduce TLL-2 activity.

Can I use recombinant TLL-2 as a positive control for Western blot with the RP-TLL2 antibody?

Yes — REC-TLL2 is the recommended positive control for the matched rabbit polyclonal antibody RP-TLL2 (/anti-tll-2-rabbit-polyclonal-antibody). Load 25–100 ng of recombinant TLL-2 per lane under reducing conditions; the antibody detects the glycosylated HEK293-expressed protein as a diffuse band at 120–135 kDa. This combination is particularly useful when validating RP-TLL2 on novel cell lysates or tissue sections, since the recombinant provides a defined, quantified reference band at the correct apparent MW. Both products are produced from the same lab platform, so epitope and species compatibility are confirmed.

How much recombinant TLL-2 should I load as a positive control for Western blot, and what antibody dilution pairs with it?

Load 50 ng of REC-TLL2 per lane as a starting point alongside your experimental samples. At this amount, RP-TLL2 used at 1:500–1:2,000 (primary, overnight 4 °C) with a standard HRP-conjugated anti-rabbit secondary at 1:5,000–1:10,000 gives a clear band at 120–135 kDa with minimal background on PVDF or nitrocellulose. If your cell lysate lane shows a faint signal, increase load to 100 ng; this titration ensures you are in the linear detection range. Total protein loaded per lysate lane should be 20–40 µg for a detectable endogenous TLL-2 band in expressing cell lines.

How should I store and handle recombinant TLL-2 to maintain activity, and what is its shelf life?

REC-TLL2 is supplied in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 5 mM CaCl2 as single-use aliquots. Store at -20 °C immediately upon receipt. Avoid repeated freeze-thaw cycles — each cycle measurably reduces metalloprotease activity due to aggregation and zinc loss. Working aliquots can be held at 4 °C for up to 48 hours; do not leave at room temperature. When diluting for assays, use the same buffer base to prevent dilution shock. Under recommended storage conditions, activity is stable for at least 12 months from the date of manufacture shown on the CoA.