Chitinase-3-Like-2 (Recombinant)

Frequently Asked Questions

What is the expected molecular weight of recombinant CHI3L2 on SDS-PAGE and Western blot?

The mature CHI3L2 sequence spans 390 residues, giving a predicted polypeptide MW of approximately 43 kDa. Because this recombinant is expressed in HEK293 cells, N-linked glycosylation is present and consistent with the native secreted form; the apparent MW on reducing SDS-PAGE typically runs at 45–50 kDa, slightly above the theoretical mass. On Western blot you may observe a diffuse or doublet band in this range reflecting glycoform heterogeneity — this is expected and not a purity concern. Purity is confirmed at >95% by SDS-PAGE.

Does recombinant CHI3L2 YKL-39 have chitinase enzymatic activity or does it cleave chitin substrates?

CHI3L2 (YKL-39/CP-39) lacks chitinase enzymatic activity despite its structural homology to glycoside hydrolase family 18 members. A critical substitution at the catalytic glutamate abolishes hydrolytic function. This recombinant will bind chitooligosaccharides and related glycan ligands with high affinity, but it will not hydrolyze chitin or fluorogenic chitin-based substrates such as 4-MU-GlcNAc2. Assay designs should therefore focus on ligand-binding, competitive displacement, or receptor-interaction readouts rather than enzymatic cleavage assays.

What assay format and substrate should I use to measure CHI3L2 glycan-binding activity in vitro?

Because CHI3L2 is a catalytically inactive chitin-binding lectin, standard chromogenic or fluorogenic hydrolysis assays are not applicable. Validated approaches include surface plasmon resonance or biolayer interferometry with immobilized chitooligosaccharides (e.g., (GlcNAc)4–(GlcNAc)6), competitive ELISA-based displacement assays, or pull-down experiments with chitin-conjugated beads. For cell-based receptor-binding studies, a starting concentration of 0.5–5 µg/mL in serum-free medium is a reasonable titration range; optimize empirically for your cell type and readout.

What buffer is recombinant Chitinase-3-Like-2 supplied in and how should I dilute it for binding assays?

This recombinant is supplied in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol. For most binding or cell-based assays, a 1:10 to 1:20 dilution into your assay buffer (e.g., PBS + 0.1% BSA, or serum-free DMEM) is a practical starting point, keeping glycerol below 0.5% final to avoid cytotoxicity or assay interference. Note that CHI3L2 does not bind heparin, so heparin-containing blocking buffers will not sequester the protein — a meaningful difference from several related family members.

How should I store recombinant CHI3L2 to maintain activity and what is the shelf life?

Store at −20°C in the single-use aliquots provided. Avoid repeated freeze-thaw cycles, which can promote aggregation and reduce glycan-binding activity. Working aliquots kept at 4°C are stable for up to 48–72 hours; for longer bench-side use, adding carrier protein (0.1% BSA) is advisable if your downstream assay permits it. Under recommended storage conditions, activity is guaranteed for 12 months from the date of receipt. Do not store in frost-free freezers subject to temperature cycling.

Can I use recombinant CHI3L2 as a positive control for Western blot with the matched TPB anti-CHI3L2 antibody RP-Chitinase3Like2?

Yes — this is one of the primary use cases for pairing REC-Chitinase3Like2 with RP-Chitinase3Like2. The rabbit polyclonal antibody was raised and validated against the same recombinant, guaranteeing immunoreactivity on Western blot. Load 20–50 ng of recombinant per lane on a standard 10–12% SDS-PAGE gel; expect a band at 45–50 kDa under reducing conditions. This also serves as a reliable positive control for antibody lot-to-lot consistency checks and for validating new detection secondaries against a defined antigen quantity.

How much recombinant CHI3L2 should I load for Western blot to establish a positive control band?

For a clean, well-resolved positive control band using RP-Chitinase3Like2, load 20–50 ng of REC-Chitinase3Like2 per lane alongside your lysate samples. At 20 ng you should see a strong signal with the polyclonal at a 1:1,000–1:2,000 primary antibody dilution; at 50 ng the band is robust enough to serve as a semi-quantitative reference even on lower-sensitivity detection systems. Because the recombinant carries native-like N-glycosylation from HEK293 expression, the band position accurately reflects what you expect from secreted endogenous CHI3L2 in conditioned medium or synovial fluid samples.

What is the isoform and processing state of this recombinant CHI3L2 — does it include the signal peptide?

This recombinant corresponds to the full-length mature form of human CHI3L2 (UniProt Q15782), encompassing all 390 residues of the processed protein. The N-terminal signal peptide (residues 1–21 by canonical annotation) is excluded, as is standard practice for secreted proteins expressed in HEK293 — the signal sequence is not present in the secreted native protein and would be cleaved co-translationally anyway. No additional pro-domains or affinity tags are present in the final product. The sequence is consistent with the predominant circulating and synovial fluid form documented in the literature.