Chitinase-3 (Recombinant)

Frequently Asked Questions

What molecular weight does recombinant Chitinase-3 / YKL-40 run at on SDS-PAGE?

The mature Chitinase-3 (CHI3L1) sequence is 378 amino acids, giving a predicted unglycosylated MW of ~42 kDa. Because this recombinant is expressed in HEK293 cells, N-linked glycosylation adds roughly 8–12 kDa, so the dominant band on reducing SDS-PAGE typically appears at 50–55 kDa. A faint higher-MW smear is occasionally visible and reflects heterogeneous glycoforms — consistent with endogenous YKL-40 behavior. If you are comparing to a deglycosylated control, PNGase F treatment collapses the band to the expected ~42 kDa core.

Does recombinant Chitinase-3 have chitinolytic enzyme activity, and how do I confirm it is active?

Human CHI3L1 lacks chitinolytic activity; critical catalytic residues present in active chitinases (most notably Glu140 in family 18) are substituted in CHI3L1, abolishing hydrolysis of the glycosidic bond. Activity is therefore assessed by chitin-oligosaccharide or heparin binding, not by 4-MU–chitotrioside fluorescence assays used for chitotriosidase or AMCase. A standard pull-down with chitin-agarose beads in 50 mM Tris-HCl pH 7.5, 150 mM NaCl serves as a functional binding confirmation. Do not use 4-methylumbelliferyl substrates — a flat fluorescence curve is expected and does not indicate a defective lot.

What assay format and binding conditions work best for Chitinase-3 heparin or ECM interaction studies?

For heparin-binding assays, a heparin-Sepharose pull-down or SPR experiment in 50 mM Tris-HCl pH 7.5, 150 mM NaCl is a reliable starting point; elution typically requires 0.5–1.0 M NaCl, consistent with published heparin-binding data for YKL-40. For ELISA-based receptor or collagen binding, coat recombinant Chitinase-3 at 0.5–2 µg/mL. Starting protein concentrations of 100–500 nM are appropriate for solution-phase binding assays. Calcium and magnesium are not required for ECM binding, and the standard storage buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol) is compatible with most assay formats after a 1:5 or greater dilution.

What is the isoform and processing state of this recombinant — does it include the signal peptide?

This recombinant corresponds to the full-length mature secreted form of human CHI3L1 (UniProt Q53FH2), with the 21-amino-acid signal peptide removed, spanning residues 22–383 of the canonical sequence (378 residues of mature protein). No propeptide cleavage is required for secretion or function. The HEK293 expression system faithfully replicates the endogenous secreted glycoprotein, making it suitable for studies where post-translational modifications — particularly N-glycosylation at Asn60 and Asn233 — are relevant to receptor binding or antibody epitope accessibility.

Can I use Chitinase-3 (Recombinant) as a positive control for Western blot with the RP-Chitinase3 antibody?

Yes — this is a primary use case. Load 20–50 ng of recombinant Chitinase-3 per lane alongside your cell lysate or conditioned medium samples. The matched rabbit polyclonal RP-Chitinase3 (/anti-chitinase-3-rabbit-polyclonal-antibody) is raised against the same recombinant, ensuring band concordance at 50–55 kDa on Western blot. This pairing eliminates the guesswork of mismatched band positions between recombinant positive control and antibody epitope. Because both products are produced from the same TPB lot pipeline, batch-to-batch consistency between the antibody and this recombinant is routinely monitored.

How much recombinant Chitinase-3 should I load for a Western blot positive control lane?

A loading range of 20–50 ng per lane is sufficient for a clear single band at 50–55 kDa using the RP-Chitinase3 rabbit polyclonal at a 1:1,000–1:2,000 dilution with standard HRP-conjugated secondary antibody and ECL detection. At 100 ng the band saturates on most film or digital imagers, which is useful for troubleshooting but not for quantitative comparisons. Dilute the recombinant stock in 1× SDS sample buffer immediately before use; do not pre-dilute in PBS and store, as glycoprotein adsorption to low-binding tubes can reduce apparent loading.

How should I handle, dilute, and store recombinant Chitinase-3 to maximize shelf life?

The protein is supplied in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol and stored at −20°C in single-use aliquots. Avoid repeated freeze-thaw cycles — activity and binding integrity decline noticeably after three or more cycles. Thaw on ice, spin briefly at 10,000 × g to collect any microdroplets, and use within 24 hours of thawing. For dilution into assay buffer, maintain at least 0.1 mg/mL BSA as a carrier if working below 10 µg/mL to prevent surface adsorption losses. Properly aliquoted and stored stocks are stable for 12 months at −20°C.

Is recombinant Chitinase-3 suitable for ELISA standard curve generation and what concentration range should I use?

This recombinant is appropriate as a calibration standard for sandwich ELISAs targeting human YKL-40/CHI3L1, provided your capture and detection antibodies recognize the glycosylated form — which the HEK293-expressed material accurately reflects. A typical standard curve spans 0.1–100 ng/mL in a matched assay diluent (e.g., 1% BSA in PBS-T). Prepare the top standard fresh from a thawed aliquot and perform serial 2- or 3-fold dilutions. Endotoxin is <0.1 EU/µg by LAL, so this recombinant is also suitable for cell-based stimulation assays where LPS contamination would confound cytokine readouts.