Anti-Cathepsin-A Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight band should I expect for Cathepsin-A on Western blot?

Full-length Cathepsin-A migrates at approximately 54 kDa corresponding to the precursor form. In lysosomes, the mature protein is proteolytically processed into a two-chain form consisting of a 32 kDa heavy chain and a 20 kDa light chain that remain associated. Depending on cell type and lysis conditions, you may observe the 54 kDa precursor, the processed chains, or both. Lysosome-enriched fractions typically show more abundant processed forms. The antibody is raised against residues spanning both chains, so it should detect precursor and mature forms, though band intensity may vary.

Does this antibody work for immunofluorescence detection of lysosomal Cathepsin-A?

Triple Point Biologics antibodies are validated for Western blot and immunohistochemistry; immunofluorescence performance has not been systematically validated for this clone. However, many researchers successfully adapt our antibodies to IF applications. For lysosomal Cathepsin-A, try starting dilutions between 1:100 and 1:500 with standard paraformaldehyde fixation and permeabilization. Include a co-stain with LAMP1 or LAMP2 to confirm lysosomal localization. Test on a known CTSA-positive cell line such as HeLa or fibroblasts before moving to experimental samples.

What is the recommended starting dilution for Western blot with this Cathepsin-A antibody?

The validated dilution is 1:1000 for Western blot. This has been tested against human lysates. Optimal dilution depends on expression level, which varies by tissue and cell type. Cathepsin-A is broadly expressed but most abundant in kidney, liver, and placenta. For initial experiments with cultured cells, start at 1:1000 in 5% non-fat milk or BSA in TBST with overnight incubation at 4°C. If signal is weak in low-expression samples, titre up to 1:500. If background is high, increase blocking time or dilute to 1:2000.

Will this antibody cross-react with mouse or rat Cathepsin-A?

The antibody is validated against human Cathepsin-A. Mouse and rat are listed as potential cross-reactive species based on sequence homology. Human CTSA shares approximately 85% identity with mouse and rat orthologs across the immunogen region. Cross-reactivity has not been experimentally confirmed in-house. If working with rodent samples, include a positive control lysate from mouse or rat tissue known to express Cathepsin-A at detectable levels, such as kidney or liver. Expect similar band patterns to human, but confirm specificity with a CTSA-deficient or knockdown control if available.

What cell lysate should I use as a positive control for Cathepsin-A expression?

HeLa, HEK293, or primary human fibroblasts provide reliable positive controls, as Cathepsin-A is constitutively expressed in lysosomes of most cell types. For tissue lysates, human placenta, kidney cortex, or liver show high endogenous expression. Load 20-30 µg total protein per lane. Lysosome-enriched fractions will yield stronger signal than whole-cell lysates. A good negative control strategy is siRNA knockdown of CTSA in your test cell line, or comparison to fibroblasts from a galactosialidosis patient if available through a repository, since these lack functional Cathepsin-A protein.

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

Store the antibody at -20°C in the original formulation. Avoid repeated freeze-thaw cycles, which can reduce titer and increase aggregation. For routine use, aliquot into single-use volumes immediately upon receipt. Working aliquots may be kept at 4°C for up to one month if sodium azide preservative is present; check the product datasheet. Do not store diluted antibody in working buffer for more than one week. Under proper storage at -20°C without freeze-thaw, polyclonal antibodies from this production lot typically remain stable for at least two years from the manufacturing date.

Does Cathepsin-A run as a single band or are there multiple isoforms to consider?

Cathepsin-A does not have alternatively spliced isoforms, but the single gene product undergoes post-translational processing. You will typically see the 54 kDa precursor and, depending on sample type, the mature processed form as separate 32 kDa and 20 kDa chains. These chains remain non-covalently associated in the native complex but may separate under reducing SDS-PAGE. If you see additional higher molecular weight bands above 54 kDa, these likely represent the multienzyme complex with beta-galactosidase and NEU1, which can survive mild lysis conditions. These complexes dissociate in standard Laemmli buffer with boiling.

Can this antibody detect the Cathepsin-A complex with beta-galactosidase and neuraminidase-1?

This antibody recognizes denatured Cathepsin-A and is validated for Western blot under reducing conditions, where the high-molecular-weight lysosomal complex dissociates. For detecting the intact approximately 1.3 MDa multienzyme complex, you would need non-denaturing methods such as Blue Native PAGE or co-immunoprecipitation followed by Western blot for individual components. If your goal is to confirm complex formation, immunoprecipitate with anti-Cathepsin-A under native conditions, then blot the precipitate for beta-galactosidase or NEU1. Standard denaturing Western blot will show only monomeric CTSA species.