Cystatin-11 (Recombinant)

Frequently Asked Questions

What molecular weight does recombinant Cystatin-11 run at on SDS-PAGE or Western blot?

Cystatin-11 has a predicted molecular weight of approximately 15.8 kDa based on its 138-amino acid sequence (UniProt Q9H912). However, because this recombinant is expressed in HEK293 cells and may carry N-linked or O-linked glycosylation, the apparent MW on SDS-PAGE typically runs slightly higher — expect a band in the 17–20 kDa range under reducing conditions. Non-reducing SDS-PAGE may shift the band further due to the disulfide-bonded cystatin fold. Always confirm against your lot-specific certificate of analysis.

Is recombinant Cystatin-11 the full-length sequence or a processed/truncated form?

This preparation uses the full-length 138-residue sequence encoded by CST11 (UniProt Q9H112), expressed in HEK293 cells with a signal peptide for secretion. The secreted, mature form after signal peptide cleavage is the predominant species recovered. Cystatin-11 is a type 2 cystatin and does not undergo propeptide removal in the same manner as some papain-family inhibitors — the inhibitory wedge loop is present in the full-length mature form. Check the certificate of analysis for confirmed N-terminal sequence of your specific lot.

Which cysteine proteases does Cystatin-11 inhibit, and what substrate should I use to measure inhibitory activity?

Cystatin-11 is a type 2 cystatin superfamily member with annotated inhibitory activity toward cathepsin-family cysteine proteases, particularly cathepsin B and cathepsin L — the canonical targets for this superfamily. To measure inhibitory activity, use the fluorogenic substrate Z-Phe-Arg-AMC (typically 10–50 µM) in a cathepsin B or cathepsin L activity assay at pH 5.5–6.0, 37°C. Pre-incubate recombinant Cystatin-11 with protease for 10–15 minutes before adding substrate. IC50 values for type 2 cystatins against cathepsin B are typically in the low nanomolar range (1–10 nM); titrate Cystatin-11 from 0.1 to 100 nM to define your IC50 empirically.

What buffer conditions are recommended for Cystatin-11 activity assays, and is the storage buffer compatible?

Recombinant Cystatin-11 is supplied in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol. For inhibitory activity assays against cathepsin B or cathepsin L, you will need to dilute into a slightly acidic assay buffer — 100 mM sodium acetate, 1 mM EDTA, 2–5 mM DTT, pH 5.5–6.0 is standard. Dilute the stock at least 1:10 into assay buffer to reduce glycerol to ≤1%, which can otherwise interfere with fluorescence readings. EDTA in the assay buffer is compatible and does not affect cystatin-family inhibitors. Avoid prolonged exposure to pH below 4.5, which may destabilize the fold.

What starting concentration of recombinant Cystatin-11 should I use in a cathepsin inhibition assay?

For an initial dose-response experiment, start with a 10-point, 3-fold serial dilution ranging from 300 nM down to approximately 0.1 nM in the assay well. This range reliably brackets the expected IC50 for type 2 cystatins against cathepsin B (typically 1–10 nM). Use equimolar or sub-equimolar protease concentrations (1–5 nM cathepsin B or L) to stay within tight-binding assay conditions. If you observe a sharp sigmoidal curve compressed into fewer than 4 dilution points, reduce protease concentration. The supplied stock concentration is indicated on your lot certificate; prepare working dilutions fresh in assay buffer on the day of use.

Can I use recombinant Cystatin-11 as a positive control for Western blot with the matched Triple Point Biologics antibody?

Yes — REC-Cystatin11 is the intended positive control for the matched rabbit polyclonal antibody (SKU: RP-Cystatin11; see /anti-cystatin-11-rabbit-polyclonal-antibody). Both the recombinant protein and the antibody are produced in the same lab and validated as a pair. Load 50–200 ng of REC-Cystatin11 per lane under reducing conditions alongside your experimental lysates. Expect a band at approximately 17–20 kDa. This combination is particularly useful when validating the antibody against a tissue lysate from epididymis or other reproductive tissue, where endogenous Cystatin-11 expression is highest.

How much recombinant Cystatin-11 should I load for Western blot positive control, and will it interfere with my lysate lanes?

50–200 ng per lane is the recommended loading range when using RP-Cystatin11 (the matched antibody, SKU: RP-Cystatin11) for Western blot detection. At 100 ng, the band is reliably detected at antibody dilutions of 1:500–1:2000 without overloading. Run the recombinant control in a dedicated lane rather than spiking into lysate; at ~17–20 kDa it is unlikely to co-migrate with most proteins of interest, but its presence in a mixed lane could complicate densitometry. Because endotoxin is <0.1 EU/µg by LAL assay, the preparation is clean enough for functional cell-based assays alongside the WB control use.

How should I store and handle recombinant Cystatin-11 to maintain activity, and what is its shelf life?

Store at -20°C in the single-use aliquots provided. The supplied buffer — 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol — is formulated to protect activity through freeze-thaw and during short-term use. Avoid repeated freeze-thaw cycles; each cycle can cause measurable loss of inhibitory activity in disulfide-bonded proteins. Once thawed, keep on ice and use within 24 hours; do not re-freeze. Shelf life at -20°C is 12 months from the date of receipt when stored correctly. If you need to dilute to working concentrations below ~10 µg/mL, add carrier protein (0.1% BSA) to the dilution buffer to reduce adsorption losses to tube surfaces.