Cystatin M (Recombinant)

Frequently Asked Questions

What molecular weight does recombinant Cystatin M run at on SDS-PAGE and Western blot?

The mature Cystatin M sequence is 149 amino acids, giving a predicted unmodified MW of ~16 kDa. Because this recombinant is produced in HEK293 cells and carries native-like N-linked glycosylation, the apparent MW on reducing SDS-PAGE typically shifts to ~20–24 kDa — consistent with published observations for the secreted glycoform. Expect a diffuse or slightly broad band in that range; treatment with PNGase F will collapse it toward the 16 kDa backbone, confirming glycan contribution. Plan your gel percentage accordingly: 12–15% polyacrylamide resolves this region cleanly.

Which proteases does recombinant Cystatin M inhibit, and what are the reported Ki values?

Cystatin M is a high-affinity inhibitor of cathepsin L (Ki ~0.07 nM), cathepsin V/L2 (Ki in the low-nanomolar range), and the asparaginyl endopeptidase legumain (Ki ~14 nM). It does not effectively inhibit cathepsin B or cathepsin S at physiological concentrations, making it useful as a selectivity probe when studying cathepsin L- or legumain-dependent proteolysis. The full 149-residue sequence in this preparation provides the complete inhibitory scaffold, including both the N-terminal trunk and the hairpin loop regions critical for tight-binding.

What activity assay substrate should I use to measure Cystatin M inhibition of cathepsin L?

For cathepsin L inhibition assays, the fluorogenic substrate Z-Phe-Arg-AMC (typically 20–50 µM in assay buffer) is the standard choice. Run cathepsin L at 1–5 nM in 50 mM sodium acetate pH 5.5, 2 mM DTT, 1 mM EDTA — pre-activating the cathepsin for 15 min before adding inhibitor. Titrate Cystatin M from ~0.01 nM to 10 nM to bracket the IC50. For legumain inhibition, substitute Z-Ala-Ala-Asn-AMC substrate at pH 5.8. A no-inhibitor control and a heat-denatured Cystatin M control should be run in parallel to confirm inhibition is activity-dependent.

What buffer conditions are recommended for Cystatin M recombinant protein activity assays?

The storage buffer — 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol — is suitable for dilution and short-term handling, but inhibition assays are typically performed at the pH optimum of the target protease (pH 5.5–6.0 for cathepsin L and legumain). Dilute Cystatin M into assay buffer immediately before use; 10% glycerol carry-over at final concentrations below 0.5% is generally well tolerated. Avoid prolonged incubation above pH 8.0 or in the absence of a stabilizing agent, as the cystatin fold can lose activity under those conditions.

What starting concentration of recombinant Cystatin M is recommended for an IC50 determination against cathepsin L?

Given cathepsin L Ki values in the 0.05–0.1 nM range for Cystatin M, an 8- to 10-point titration spanning 0.005–50 nM Cystatin M at a fixed cathepsin L concentration of ~2 nM (near Km for substrate) is a practical starting range. Keep the inhibitor:enzyme molar ratio ≥10 at the top of the curve to ensure saturation. Protein concentration in the stock should be verified by A280 or BCA assay before setting up dilutions, as accurate inhibitor concentration is the largest source of IC50 variability in tight-binding systems like this one.

Can I use recombinant Cystatin M as a positive control for Western blot with the matched TPB antibody?

Yes — this is one of the primary applications. The matched rabbit polyclonal antibody (SKU: RP-CystatinM) was raised and validated against the same Cystatin M sequence, and the recombinant protein is the most reliable positive control for confirming antibody performance. Load 50–200 ng of recombinant Cystatin M per lane on a 12–15% SDS-PAGE gel alongside your cell lysate. The antibody detects the glycosylated band at ~20–24 kDa. Because both products originate from the same production program, this pairing also serves as a clean benchmark for antibody lot-to-lot consistency checks.

How much recombinant Cystatin M should I load for a Western blot positive control lane?

50–200 ng per lane is the recommended starting range when using RP-CystatinM at a 1:500–1:2000 primary antibody dilution. Start with 100 ng if you have no prior baseline for your detection system (ECL vs. fluorescent secondary, film vs. digital imager). The glycosylated band at ~20–24 kDa should be clearly resolved. If you observe signal saturation, drop to 25–50 ng; if signal is weak, increase to 200 ng before adjusting antibody concentration. Reducing conditions (β-mercaptoethanol or DTT in sample buffer) are appropriate — Cystatin M has a single disulfide bridge but runs well under standard reducing SDS-PAGE.

How should I store and handle recombinant Cystatin M to maintain activity over time?

Upon receipt, store at -20°C in the single-use aliquots as supplied. The storage buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol) provides adequate cryoprotection at -20°C for at least 12 months from the date of manufacture. Avoid repeated freeze-thaw cycles — each cycle risks aggregation and loss of inhibitory activity, which is particularly problematic for tight-binding inhibitors where even small activity losses distort Ki measurements. For working dilutions, prepare fresh on the day of the assay in assay-compatible buffer, and keep on ice. Do not store diluted protein for re-use.