Anti-Cathepsin-L Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight bands should I expect for Cathepsin-L on Western blot?

Cathepsin-L is synthesized as a ~38-39 kDa preproenzyme, processed to a ~37 kDa proenzyme (procathepsin L), and matured through a single-chain intermediate (~30 kDa) into the active mature form, which appears as a heavy chain around 24-25 kDa and, depending on processing, a light chain around 5 kDa. In most lysates you will see a combination of the ~37 kDa proform and the ~25 kDa mature heavy chain. Secreted procathepsin L in conditioned media typically runs at 37-39 kDa due to mannose-6-phosphate glycosylation. Treat band pattern as a readout of processing state.

How do I distinguish Cathepsin-L from Cathepsin-V (CTSL2) on a blot?

Cathepsin-V (CTSL2/CTSL3) shares ~78% sequence identity with Cathepsin-L and runs at a similar molecular weight (mature form ~26 kDa). Polyclonal reagents raised against full-length or large fragments of CTSL can show cross-reactivity with CTSL2. If your tissue is thymus, testis, or cornea — where CTSL2 is enriched — include a CTSL2 knockdown or recombinant CTSL2 control to confirm specificity. siRNA knockdown of CTSL in your cell line of interest remains the most reliable specificity control. Cathepsin-B and -S run at distinct sizes and are not a typical confounder.

What dilution should I start with for Western blot?

Start at 1/1000 in 5% non-fat milk or BSA in TBST, with overnight incubation at 4°C. For lysates with low endogenous Cathepsin-L (e.g., non-secretory cell lines), titrate up to 1/500. For high-expressing samples such as macrophages, hepatocytes, or tumor lysates, 1/2000 to 1/5000 may give cleaner signal. Load 20-30 µg total protein. Because the proform and mature form differ in abundance and processing, run gels long enough to resolve the 24-37 kDa range — a 12-15% gel is recommended.

What are good positive control lysates for Cathepsin-L?

HeLa, HEK293, A549, and HepG2 express detectable endogenous Cathepsin-L and are reliable positive controls. Macrophage lines (RAW 264.7 in mouse studies; THP-1 differentiated with PMA in human) show strong expression of both pro- and mature forms. Liver, kidney, and spleen tissue lysates are robust tissue-level controls. For secreted procathepsin L, concentrated conditioned medium from serum-starved fibroblasts or transformed cells (e.g., MDA-MB-231) works well. Pair with a CTSL siRNA or CRISPR knockout lysate as a negative control to confirm band identity.

How should I prepare samples to preserve Cathepsin-L processing intermediates?

Cathepsin-L is autocatalytic and active at acidic pH, so lysates can self-digest during preparation. Lyse on ice in a neutral-pH buffer (e.g., 50 mM Tris pH 7.4, 150 mM NaCl, 1% Triton X-100) supplemented with E-64 (10 µM) or leupeptin (10 µg/mL) to inhibit cysteine protease activity, plus a broad protease inhibitor cocktail. Avoid acidic lysis buffers unless you are intentionally activating the enzyme. Boil samples in Laemmli buffer immediately. For secreted procathepsin L analysis, collect serum-free conditioned media and concentrate with TCA or 10 kDa MWCO filters.

Is this antibody validated for mouse or rat Cathepsin-L?

This lot is validated on human samples. Human CTSL shares roughly 75% identity with mouse Ctsl and 76% with rat Ctsl at the protein level, so cross-reactivity is plausible but not guaranteed depending on the epitope region. We list mouse and rat as predicted only. If you intend to use this reagent on rodent samples, run a pilot blot with a known positive tissue (mouse liver or spleen) alongside a CTSL knockout or knockdown control. Report back to us — we update reactivity claims based on customer-validated data.

Can I use this antibody for immunoprecipitation or activity-based profiling?

This antibody is validated for Western blot. It has not been formally validated for IP. Polyclonal rabbit reagents against Cathepsin-L often work for native IP because they recognize multiple epitopes, but pilot the application with 2-5 µg per mg lysate and confirm pulldown by blotting with an orthogonal antibody. For activity studies, this antibody detects total enzyme (pro + mature) and does not discriminate active from inactive species — pair with an activity-based probe such as DCG-04 if you need to quantify active Cathepsin-L.

How should I store the antibody and how stable is it?

Store at -20°C upon receipt. The antibody is supplied in PBS with 0.02% sodium azide and 50% glycerol, which keeps it liquid at -20°C and allows direct pipetting without a freeze-thaw cycle. For long-term storage beyond 12 months, aliquot and hold at -80°C. Avoid repeated freeze-thaw cycles — more than 5 cycles can degrade signal, particularly for IF applications. Do not store at 4°C beyond 1-2 weeks. If you observe precipitate, spin at 10,000 × g for 2 minutes and use the supernatant; activity is typically unaffected.