Cystatin-9-Like-1 (Recombinant)

Frequently Asked Questions

What molecular weight should I expect for recombinant Cystatin-9-Like-1 on SDS-PAGE or Western blot?

The full-length 145-residue sequence predicts a theoretical MW of approximately 16–17 kDa. On reducing SDS-PAGE, the recombinant typically migrates at 18–22 kDa due to the HEK293 expression system, which can introduce N-linked glycosylation and other post-translational modifications common to secreted extracellular proteins. Purity is confirmed at >95% by SDS-PAGE before release. When running a Western blot positive control alongside RP-Cystatin9Like1, expect the primary band in the 18–22 kDa range; apparent MW may shift slightly under non-reducing conditions if intramolecular disulfide bonds alter protein compaction.

Does Cystatin-9-Like-1 have a signal peptide or propeptide that is cleaved, and which form does this recombinant represent?

Like other type 2 cystatins, Cystatin-9-Like-1 (UniProt Q9H114) contains an N-terminal signal peptide that is cleaved during secretion. This recombinant is expressed in HEK293 cells, which process the signal peptide co-translationally, yielding the mature secreted form of the protein. The full 1–145 residue sequence is encoded in the construct, so the final active protein corresponds to the signal-peptide-processed, extracellularly relevant form. Researchers planning structural or inhibition studies should note they are working with the mature, biologically processed species, not a cytoplasmic or pro-form artifact.

Is Cystatin-9-Like-1 a functional cysteine protease inhibitor, and which papain-family enzymes should I test it against?

Cystatin-9-Like-1 belongs to the Cres/Testatin subgroup of type 2 cystatins, a clade distinguished from classical inhibitory cystatins by divergent structural features. Canonical type 2 cystatins inhibit papain-family cysteine proteases (cathepsins B, H, L, and S are the standard panel). Whether Cystatin-9-Like-1 retains full inhibitory activity against these targets or has attenuated/redirected function — consistent with other Cres subgroup members — should be evaluated empirically. We recommend starting inhibition assays with cathepsin L (fluorogenic substrate Z-Phe-Arg-AMC, Ex/Em 380/460 nm) as the most sensitive readout, using a pre-incubation of recombinant protein with enzyme before substrate addition.

What activity assay format and substrate work best for testing Cystatin-9-Like-1 inhibitory function?

A standard fluorescence-based inhibition assay is the most practical first approach. Incubate cathepsin L (or cathepsin B as a secondary target) with serial dilutions of Cystatin-9-Like-1 in 50 mM sodium acetate pH 5.5, 1 mM EDTA, 1 mM DTT for 10–15 minutes at 37°C before adding Z-Phe-Arg-AMC (final 10–50 µM). Measure AMC release at Ex 380 nm / Em 460 nm. Include a no-inhibitor control and a known inhibitor (E-64) as a positive inhibition control. Note that the Cres subgroup may show reduced or substrate-selective inhibition; include cathepsin H and S for a broader inhibitory profile assessment.

What starting concentration of recombinant Cystatin-9-Like-1 should I use for an IC50 determination against cathepsin L?

For IC50 determination, a 10-point serial dilution covering 0.1 nM to 1 µM of Cystatin-9-Like-1 is a reasonable starting range, given that classical type 2 cystatins typically inhibit cathepsin L with Ki values in the low nanomolar range (1–10 nM). However, Cres-subgroup members may have substantially higher Ki values; running a preliminary single-concentration screen at 10 nM, 100 nM, and 1 µM against a fixed enzyme concentration (2–5 nM cathepsin L, ~20% substrate turnover at baseline) will bracket the active range before committing to a full curve.

What buffer is this recombinant Cystatin-9-Like-1 supplied in, and is it compatible with standard cysteine protease assay buffers?

Recombinant Cystatin-9-Like-1 is supplied in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol. This storage buffer is not directly compatible with most cathepsin activity assay buffers (typically sodium acetate pH 5.0–5.5 with DTT and EDTA), so a buffer exchange or high dilution into assay buffer is necessary. A minimum 1:10 dilution into assay buffer will reduce glycerol to ≤1%, which is generally tolerated. For sensitive assays, desalt via spin column (10 kDa MWCO) into assay buffer immediately before use. Avoid adding DTT to the recombinant stock directly, as reducing agents may disrupt native disulfide bonds.

Can I use recombinant Cystatin-9-Like-1 as a Western blot positive control for the matched RP-Cystatin9Like1 rabbit polyclonal antibody?

Yes — this is one of the primary intended uses of the pairing. REC-Cystatin9Like1 and RP-Cystatin9Like1 are developed in-house from the same antigen, and compatibility for Western blot is confirmed. Load 20–50 ng of recombinant per lane on a 12–15% SDS-PAGE gel; the band should appear at 18–22 kDa under reducing conditions. Run alongside your experimental lysate lanes so the positive control band serves as both a size marker and a signal-intensity reference. For antibody titration experiments, the recombinant also provides a defined-concentration standard for quantitative Western blot calibration.

How should I handle, store, and aliquot recombinant Cystatin-9-Like-1 to maintain activity over time?

Upon receipt, briefly centrifuge the vial, then aliquot into single-use volumes matching your typical assay requirements (5–20 µg aliquots are practical for most inhibition or Western experiments). Store aliquots at -20°C; the 10% glycerol in the storage buffer provides cryoprotection. Avoid repeated freeze-thaw cycles — each cycle risks aggregation and activity loss in secreted disulfide-containing proteins. Working aliquots can be held at 4°C for up to one week. Do not dilute the entire stock; prepare fresh working dilutions from a frozen aliquot immediately before each experiment to ensure reproducible starting activity.