Anti-Chitinase-3-Like-1 Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight should I expect for CHI3L1/YKL-40 on Western blot?

CHI3L1 migrates at approximately 40 kDa on reducing SDS-PAGE, though the calculated molecular weight from the 383-amino acid sequence is 42.6 kDa. The protein is glycosylated at multiple sites, which can shift the apparent mass slightly depending on post-translational modification status and cell type. Some laboratories report bands between 38-42 kDa. The YKL-40 designation itself refers to the tyrosine-lysine-leucine N-terminal residues and 40 kDa mass. If you observe higher molecular weight species, consider that CHI3L1 can form oligomers under non-reducing conditions or in certain buffer systems.

Does this antibody work for mouse or rat CHI3L1 detection?

This antibody is validated for human CHI3L1. Cross-reactivity with mouse or rat orthologs has not been experimentally confirmed. Human CHI3L1 shares approximately 60-65% sequence identity with mouse Chi3l1 (also called BRP-39) and rat orthologs, which limits reliable prediction of cross-reactivity. If you require detection in rodent samples, we recommend confirming reactivity in your system with appropriate positive controls or considering antibodies raised specifically against the mouse or rat sequences. Species-specific validation data is available upon request.

What dilution should I start with for Western blot of CHI3L1?

Start at 1:1000 dilution in blocking buffer for Western blot, as recommended in our validation data. This concentration has been optimized for detection of CHI3L1 from human cell lysates and conditioned media. Because CHI3L1 is a secreted protein, expect stronger signal in conditioned medium or serum samples compared to whole-cell lysates. If background is high, titrate up to 1:2000; if signal is weak with low-abundance samples, try 1:500. Incubation overnight at 4°C often improves sensitivity compared to one-hour room-temperature binding.

Is CHI3L1 an intracellular or secreted protein for sample preparation?

CHI3L1 is a secreted glycoprotein, so it is most abundant in conditioned culture medium, serum, plasma, or extracellular tissue fractions. For Western blot, concentrate conditioned medium 10-20 fold using centrifugal filters (10 kDa cutoff) to improve detection. Whole-cell lysates will contain primarily intracellular precursor and Golgi-resident forms. If analyzing tissue, consider preparing separate cytosolic and secreted/extracellular fractions. Avoid repeated freeze-thaw cycles of serum or medium samples, as CHI3L1 stability can decrease. For IHC, extracellular and perinuclear Golgi staining patterns are typical.

What are good positive and negative controls for CHI3L1 antibody validation?

Human macrophages, particularly M2-polarized macrophages, are strong positive controls as they secrete high levels of CHI3L1. Chondrocytes, synovial fibroblasts, and certain cancer cell lines (e.g., U87 glioblastoma, MDA-MB-231 breast cancer) also express CHI3L1 constitutively or upon IL-13 or IL-6 stimulation. For negative controls, use lysates from CHI3L1 knockout cells if available, or unstimulated fibroblasts and epithelial lines with low basal expression. Including recombinant human CHI3L1 protein as a lane control confirms the expected 40 kDa band. Peptide competition assays can further verify specificity.

Does CHI3L1 have splice variants or isoforms I should be aware of?

The primary CHI3L1 transcript encodes a single 383-amino acid protein with a 21-residue signal peptide. Alternative splicing has not been widely reported for CHI3L1, and most studies focus on the full-length secreted form. However, differential glycosylation across tissues and disease states can produce minor mobility shifts on gels. In some inflammatory contexts, proteolytic cleavage products or degradation fragments may appear as lower molecular weight bands. If you observe multiple bands, verify identity by peptide competition or mass spectrometry. The antibody targets a broad epitope region, so it should detect the predominant full-length form.

Can I use this CHI3L1 antibody for immunohistochemistry or immunofluorescence?

Yes, Triple Point Biologics antibodies are validated for Western blot; IHC and immunofluorescence validation is in progress. For IHC on formalin-fixed paraffin-embedded tissue, antigen retrieval is typically required; citrate buffer (pH 6.0) heat-induced epitope retrieval is a standard starting point. Expect extracellular and cytoplasmic perinuclear staining in CHI3L1-expressing cells such as macrophages, chondrocytes, and activated fibroblasts. Start with a 1:100-1:200 dilution for IHC and titrate based on signal intensity and background. For IF, 1:50-1:100 is a common range. Include isotype controls to assess non-specific binding.

How should I store the CHI3L1 antibody and what is the shelf life?

Store the antibody at -20°C in small aliquots to avoid repeated freeze-thaw cycles, which can reduce titer and increase aggregation. The antibody is supplied in a stabilized buffer and remains stable for at least 12 months when stored properly at -20°C. For frequent use, keep a working aliquot at 4°C for up to one month; add 0.02-0.05% sodium azide as preservative if desired, though avoid azide if using the antibody for applications involving HRP-conjugated secondaries. Do not store diluted antibody for extended periods; prepare fresh working dilutions in blocking buffer for each experiment.