Anti-Chitinase-3-Like-2 Rabbit Polyclonal Antibody

Frequently Asked Questions

What is the expected molecular weight for CHI3L2 on Western blot?

CHI3L2 migrates at approximately 39-42 kDa on reducing SDS-PAGE, slightly above its predicted molecular weight of 42.6 kDa for the mature protein. The 390-amino acid translation product includes a signal peptide (residues 1-21) that is cleaved during secretion, yielding a mature 369-residue polypeptide. You may observe broader or higher apparent molecular weight due to N-linked glycosylation at multiple sites. Deglycosylation with PNGase F can confirm glycan contribution. The protein is also known as YKL-39 or Chondrocyte protein 39, reflecting its original identification as a 39 kDa chondrocyte secretory product.

What dilution should I start with for Western blot detection of CHI3L2?

We recommend starting at 1:1000 dilution for Western blot, which has been validated with this antibody. Optimal dilution depends on your sample type and CHI3L2 expression level. Chondrocytes and osteoarthritic cartilage lysates typically yield strong signal at 1:1000 to 1:2000. For samples with lower expression, such as certain macrophage or tumor cell lines, you may need 1:500. Because CHI3L2 is a secreted protein, conditioned media or synovial fluid often requires concentration (10-20-fold by ultrafiltration) before loading. Include a dilution series in your first experiment to establish the linear detection range for your sample matrix.

Does this CHI3L2 antibody cross-react with other chitinase-like proteins?

CHI3L2 shares significant sequence homology with CHI3L1 (YKL-40, approximately 50% identity) and more distant similarity to other family members including CHIT1 and acidic mammalian chitinase. This polyclonal antibody is validated for human CHI3L2 specificity, but cross-reactivity with CHI3L1 should be assessed in your experimental system, particularly when analyzing samples that co-express both proteins, such as synovial tissue or certain tumor microenvironments. Running recombinant CHI3L1 alongside CHI3L2 as a specificity control is advisable for samples where both proteins may be present. Knockout or knockdown lysates provide the most definitive specificity confirmation.

Is this antibody likely to detect mouse or rat CHI3L2?

The antibody is validated for human CHI3L2 and has not been tested against mouse or rat orthologs. Human CHI3L2 shares approximately 65-70% sequence identity with rodent orthologs, so cross-reactivity is possible but not guaranteed. If you are working with mouse or rat samples, we recommend testing the antibody alongside a positive control (human CHI3L2 lysate or recombinant protein) and confirming band identity by pre-incubation with blocking peptide if available, or by comparing wild-type and knockout tissue. Rat and mouse each express multiple chitinase-like proteins, so specificity should be verified carefully in rodent models.

What positive control samples should I use for CHI3L2 Western blots?

Primary human chondrocytes or chondrocyte cell lines such as C-28/I2 serve as reliable positive controls, as CHI3L2 is abundantly expressed in cartilage. Osteoarthritic cartilage explant lysates or synovial tissue from OA patients also express high levels. For secreted CHI3L2, collect conditioned medium from cultured chondrocytes after 48-72 hours in serum-free conditions and concentrate 10-20-fold. Recombinant human CHI3L2 protein provides a defined control to confirm antibody reactivity and establish expected molecular weight. Some macrophage populations and tumor cell lines express CHI3L2, but levels vary widely, so verify expression by qPCR or published data before relying on these as controls.

Can I use this antibody for immunohistochemistry on cartilage tissue?

This antibody is suitable for IHC applications, which are validated across the Triple Point Biologics antibody line. For cartilage tissue, antigen retrieval is typically required due to the dense extracellular matrix; citrate buffer (pH 6.0) heat-mediated retrieval for 20 minutes is a reasonable starting point. CHI3L2 localizes primarily to chondrocytes in the pericellular and territorial matrix regions. In osteoarthritic cartilage, expression is elevated and more widespread. Blocking endogenous peroxidase and using appropriate species-matched blocking sera (this is a rabbit polyclonal) are essential. Synovial tissue sections from OA or RA patients also serve as positive controls, showing staining in synovial fibroblasts and infiltrating macrophages.

How should I prepare cell lysates to detect secreted CHI3L2?

Because CHI3L2 is a secreted glycoprotein, you will detect minimal intracellular protein in whole-cell lysates under steady-state conditions. To capture secreted CHI3L2, collect conditioned medium from cultured cells after 24-72 hours. Switch to serum-free or low-serum medium to reduce albumin background, but verify that cells remain viable. Concentrate conditioned medium 10-20-fold using centrifugal filters (10 kDa MWCO) before mixing with sample buffer. For intracellular detection, lysates from cells treated with brefeldin A or monensin to block secretion can retain CHI3L2, or examine chondrocytes undergoing active synthesis. Tissue lysates from cartilage or synovium should include both cellular and extracellular matrix fractions for comprehensive detection.

What are the recommended storage conditions for this CHI3L2 antibody?

Store the antibody at -20°C in small aliquots to avoid repeated freeze-thaw cycles, which can reduce titer and increase aggregate formation. The antibody is supplied as a rabbit polyclonal at 100 µg per vial. If you use the antibody frequently, a working aliquot can be kept at 4°C for up to one month with 0.02-0.05% sodium azide as preservative, though long-term storage at -20°C is preferable. Avoid storing diluted antibody; prepare fresh working dilutions in blocking buffer for each experiment. After thawing, mix gently by inversion rather than vortexing to minimize denaturation. Centrifuge briefly before use if any precipitate is visible.