Chitinase-3-Like-1 (Recombinant)

Frequently Asked Questions

What molecular weight does recombinant CHI3L1 / YKL-40 run at on SDS-PAGE?

The unglycosylated mature CHI3L1 sequence predicts ~40 kDa, but because this recombinant is expressed in HEK293 cells and carries native-like N-glycosylation, it typically migrates at 42–45 kDa under reducing SDS-PAGE conditions. Glycosylation heterogeneity can produce a slightly diffuse band; this is expected and not a sign of degradation. Our lot-release SDS-PAGE confirms >90% purity at this position. If you are running a denaturing Western blot with RP-Chitinase3Like1, load 20–50 ng per lane and expect the band in the 42–45 kDa window.

Does recombinant CHI3L1 have chitinase enzymatic activity or is it catalytically inactive?

CHI3L1 is catalytically inactive despite belonging to glycoside hydrolase family 18. A glutamate residue critical for hydrolysis in true chitinases is substituted in CHI3L1, abolishing glycosidic bond cleavage. This recombinant therefore will not hydrolyze 4-methylumbelliferyl-β-D-N,N′-diacetylchitobioside (4-MU-chitobiose) or similar fluorogenic chitinase substrates. Its utility lies in chitin-binding affinity assays, heparin-binding studies, receptor interaction experiments (e.g., IL-13Rα2, CRTH2), and as a quantitative ELISA standard — not in enzymatic activity readouts.

What substrate or binding assay should I use to confirm recombinant CHI3L1 is functional?

Because CHI3L1 lacks hydrolytic activity, functionality is best confirmed by chitin-bead pull-down or surface plasmon resonance (SPR) against chitin oligosaccharides (e.g., (GlcNAc)₄–(GlcNAc)₆). Heparin-affinity chromatography retention is a complementary assay — CHI3L1 binds heparin with reported Kd in the low-nanomolar range. For cell-based work, dose-dependent induction of IL-8 or MMP production in fibroblasts (starting at 100–500 ng/mL) serves as a functional readout. A negative result in a 4-MU-based chitinase assay is, paradoxically, also consistent with intact recombinant identity.

What buffer and storage conditions are recommended for recombinant CHI3L1 long-term stability?

This recombinant is supplied in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol. Aliquot immediately upon receipt and store at −20 °C; avoid repeated freeze-thaw cycles, which can cause aggregation and reduce binding activity. For working dilutions, PBS or HEPES-buffered saline (pH 7.2–7.4) supplemented with 0.1% BSA is suitable. The protein is stable at 4 °C for up to 72 hours after thawing. Do not dilute below ~10 µg/mL in carrier-free buffer without BSA supplementation, as surface adsorption losses become significant at sub-microgram concentrations.

What starting concentration of recombinant CHI3L1 should I use for a YKL-40 ELISA standard curve?

For a standard ELISA sandwich assay, a top standard of 10–20 ng/mL with 2-fold serial dilutions down to ~0.156 ng/mL covers the typical serum and synovial fluid dynamic range reported in the literature. Prepare standards in the same matrix used for unknowns (e.g., 1% BSA in PBS for buffer-based assays, or charcoal-stripped serum for clinical samples) to minimize matrix mismatch. Stock concentration should be verified by absorbance at 280 nm (ε calculated from sequence) or BCA assay before generating the curve. Lot-to-lot CV on our release standards is ≤8%.

Can I use recombinant CHI3L1 as a Western blot positive control with the RP-Chitinase3Like1 antibody?

Yes — this recombinant was validated specifically alongside RP-Chitinase3Like1 (/anti-chitinase-3-like-1-rabbit-polyclonal-antibody) for this purpose. Load 20–50 ng of recombinant per lane on a 10–12% SDS-PAGE gel under reducing conditions; the antibody detects a clean band at 42–45 kDa. Use RP-Chitinase3Like1 at 1:1,000–1:2,000 dilution with standard HRP-conjugated anti-rabbit secondary. This pairing is also useful for antibody lot validation: consistent signal intensity and band position across recombinant lots confirms antibody performance is stable.

How much recombinant CHI3L1 should I load to optimize a positive control lane for RP-Chitinase3Like1 immunoblot?

20 ng per lane is sufficient for a clean, non-saturating signal with RP-Chitinase3Like1 at 1:1,000 dilution using standard ECL detection. If you are using a highly sensitive substrate (e.g., femtogram-range ECL), reduce loading to 5–10 ng to avoid signal saturation. Run the recombinant lane adjacent to your cell lysate to confirm co-migration at 42–45 kDa; note that endogenous CHI3L1 in lysates from high-expressing lines (e.g., MDA-MB-231, U87) may appear slightly higher due to differential glycosylation. The recombinant band remains the most reliable MW reference for this target.

What dose range of recombinant CHI3L1 is appropriate for dose-response cell stimulation experiments?

Published dose-response studies report detectable downstream signaling (AKT, ERK phosphorylation; IL-8 secretion) starting at 50–100 ng/mL, with maximal or near-maximal effects typically observed at 500–1,000 ng/mL in fibroblast and macrophage models. A practical starting series is 0, 50, 100, 250, 500, and 1,000 ng/mL in complete media. The recombinant's endotoxin level is <0.1 EU/µg by LAL, so cytokine responses at these concentrations are attributable to CHI3L1 activity rather than LPS contamination — an important control point to document in the methods. Include a heat-inactivated aliquot as negative control.