Anti-Chymase Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight should I expect for Chymase on a Western blot?

Chymase (CMA1) migrates at approximately 30 kDa on reducing SDS-PAGE, corresponding to the mature secreted form after signal peptide cleavage. The full-length precursor is 247 residues, but the 26-residue N-terminal signal sequence is removed during secretion from mast cells. Some samples may show minor higher-molecular-weight bands around 37 kDa representing glycosylated forms or incompletely processed enzyme. If working with mast cell lysates or granule preparations, the 30 kDa band should be prominent. Recombinant Chymase standards typically run at the same 30 kDa position.

What starting dilution should I use for Chymase Western blots?

Start at 1:1000 dilution for Western blot with this rabbit polyclonal antibody, as recommended from our validation data. Optimal dilution depends on Chymase expression level in your sample; mast cell-rich tissues like skin, lung, or heart will yield strong signal at 1:1000, while samples with sparse mast cell populations may require 1:500. Incubate overnight at 4°C for best results. If background is high, titrate up to 1:2000. Always include a positive control lysate from human mast cell lines (HMC-1, LAD2) or mast cell-enriched tissue to confirm antibody performance.

Does this antibody cross-react with other chymotryptic proteases like cathepsin G or mast cell tryptase?

Chymase shares structural similarity with other serine proteases, but this antibody was raised against Chymase-specific epitopes. We have not observed cross-reactivity with cathepsin G (approximately 29 kDa) or mast cell tryptase (approximately 35 kDa) in validated human samples. However, because multiple chymotrypsin-fold proteases exist in mast cell granules, we recommend running positive controls for Chymase alongside your experimental samples. If you observe unexpected bands, consider siRNA knockdown or comparison with Chymase-deficient cell lines to confirm specificity in your particular tissue context.

Will this antibody work with mouse or rat samples?

This antibody is validated for human Chymase (CMA1). Reactivity with mouse or rat Chymase is not validated. Mouse expresses multiple mast cell chymases (mMCP-1, -2, -4, -5, -9) with varying homology to human CMA1, and rats express rMCP-1, -2, and -5. Sequence identity between human CMA1 and rodent orthologs ranges from 65-75 percent, so cross-reactivity is possible but not assured. If you need to work with rodent samples, we recommend testing at multiple dilutions with species-appropriate positive controls or considering a rodent-specific Chymase antibody.

What positive control tissue should I use for Chymase immunohistochemistry?

Human skin, lung parenchyma, or myocardium provide reliable positive controls for Chymase IHC because these tissues contain abundant resident mast cells. In normal skin, Chymase-positive mast cells appear in the papillary and reticular dermis with characteristic cytoplasmic granular staining. Tonsil and gastrointestinal mucosa also work well. For disease models, fibrotic tissue or sites of allergic inflammation show elevated mast cell numbers. Perform antigen retrieval with citrate buffer (pH 6.0) for formalin-fixed paraffin-embedded sections. Negative controls should include Chymase-poor tissues like skeletal muscle or omission of primary antibody.

Should I add protease inhibitors when preparing lysates for Chymase Western blot?

Yes, include a broad-spectrum protease inhibitor cocktail when lysing cells or tissues for Chymase detection. Chymase itself is a potent serine protease, and upon cell lysis it can be released from secretory granules and potentially degrade other proteins or undergo autocatalytic processing. Standard serine protease inhibitors like PMSF or AEBSF are advisable, though they may not completely inhibit Chymase activity at typical concentrations. Work quickly on ice, and snap-freeze aliquots to minimize degradation. Avoid repeated freeze-thaw cycles, which can disrupt mast cell granules and release active enzyme into the lysate.

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

Store the antibody at -20°C in the supplied buffer. As a rabbit polyclonal, it remains stable for at least one year under these conditions. Avoid repeated freeze-thaw cycles by making single-use aliquots upon receipt; 5-10 µL aliquots work well for most Western blot experiments given the 1:1000 recommended dilution. If you need to store diluted antibody, add 0.02 percent sodium azide and keep at 4°C for up to one month, though we generally recommend preparing fresh working dilutions. Do not store diluted antibody without preservative, as bacterial contamination will degrade immunoglobulin over time.

Can I detect secreted Chymase in cell culture supernatants or is it only intracellular?

Chymase is stored in mast cell secretory granules and released upon degranulation, so it can be detected in culture supernatants after appropriate stimulation. Treat mast cells with calcium ionophore (A23187), IgE cross-linking, or compound 48/80 to trigger degranulation, then collect supernatants after 30-60 minutes. Concentrate supernatants 10-20 fold using centrifugal filters (10 kDa cutoff) before Western blot, as secreted concentrations may be low. Note that released Chymase retains enzymatic activity and may degrade matrix proteins in serum-containing media; work quickly and add protease inhibitors to supernatants immediately after collection.