Cystatin Like 1 (Recombinant)

Frequently Asked Questions

What is the expected molecular weight of Cystatin Like 1 (Recombinant) on SDS-PAGE?

The full-length CSTL1 sequence encodes 145 amino acids, giving a predicted unmodified MW of approximately 16.5 kDa. Because this recombinant is produced in HEK293 cells — which support N-linked glycosylation and other secretory pathway modifications — the apparent MW on reducing SDS-PAGE typically runs slightly higher, in the 18–22 kDa range depending on gel percentage and run conditions. Use a 12–15% polyacrylamide gel for cleanest resolution. Purity is confirmed >90% by SDS-PAGE prior to release.

Is Cystatin Like 1 processed or cleaved in the secretory pathway, and does the recombinant reflect the mature form?

CSTL1 carries a predicted signal peptide (approximately residues 1–20) that directs it into the secretory pathway. In HEK293 expression, the signal peptide is cleaved co-translationally, so the secreted recombinant protein represents the mature processed form beginning around residue 21. Full-length sequence coverage of all 145 residues is used in the construct design, ensuring the signal sequence is present for correct processing. Researchers working with tissue-derived CSTL1 can therefore use this recombinant as a direct molecular weight and immunoreactivity reference.

Does Cystatin Like 1 inhibit cysteine proteases like papain or cathepsin B, and what assay substrate should I use?

Like other type 2 cystatin superfamily members, CSTL1 is studied as a candidate cysteine protease inhibitor, though inhibitory potency in the Cres/Testatin subgroup is generally weaker or more context-dependent than canonical cystatins such as cystatin C. For benchmarking inhibitory activity, a standard fluorogenic substrate assay using Z-Phe-Arg-AMC (excitation 355 nm / emission 460 nm) with papain or cathepsin B at pH 6.0–6.5 in 100 mM sodium phosphate, 1 mM EDTA, 1 mM DTT is a practical starting point. Include a canonical cystatin C recombinant as a positive inhibition control to validate assay sensitivity before interpreting CSTL1-specific results.

What starting concentration of Cystatin Like 1 (Recombinant) should I use for a protease inhibition assay?

For initial dose-response experiments, test CSTL1 across a range of 0.1–10 µM against a fixed protease concentration (typically 2–5 nM papain or cathepsin B). Given that the Cres/Testatin subgroup can exhibit reduced inhibitory constants relative to canonical cystatins, err toward the higher end of this range for first-pass IC50 estimation. Dilute from the stock into assay buffer (100 mM sodium phosphate pH 6.0–6.5, 1 mM EDTA, 1 mM DTT) rather than directly into the storage buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol), as the glycerol content can affect fluorescence baselines at high concentrations.

How do I optimize buffer conditions when using Cystatin Like 1 (Recombinant) in a cysteine protease inhibition assay?

The storage buffer — 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol — is formulated for protein stability, not assay compatibility. For cysteine protease assays, keep the final glycerol contribution below 1% (v/v) by diluting at least 1:10 into assay buffer before adding to the reaction. Ensure DTT or TCEP (1–2 mM) is present to maintain protease active-site cysteines in a reduced state. If working at physiological pH (7.4), note that cathepsin B activity decreases substantially above pH 7.0; papain remains more active and is the preferred enzyme for neutral-pH inhibition screens.

Can I use Cystatin Like 1 (Recombinant) as a positive control for Western blot with the matched rabbit polyclonal antibody?

Yes — this is one of the primary use cases for REC-CystatinLike1. The matched antibody (SKU: RP-CystatinLike1; see /anti-cystatin-like-1-rabbit-polyclonal-antibody) is produced and validated in the same lab against CSTL1, and compatibility with this recombinant for Western blot is confirmed. Load 50–100 ng of recombinant protein per lane alongside your cell or tissue lysate. Expect a band in the 18–22 kDa range under reducing conditions. This side-by-side loading lets you confirm antibody sensitivity and confirm that a band in your lysate co-migrates with the correctly processed recombinant form.

How much recombinant Cystatin Like 1 should I load for Western blot positive control, and what antibody dilution works?

Load 50–100 ng per lane for a strong, clean signal with RP-CystatinLike1 on standard PVDF or nitrocellulose membranes. If you are titrating antibody concentration, 25 ng can still produce a detectable band at higher antibody concentrations. For the RP-CystatinLike1 rabbit polyclonal antibody (SKU: RP-CystatinLike1), a starting dilution of 1:500–1:2000 in 5% non-fat dry milk/TBST is a reasonable range; optimize for your specific lysate background. The recombinant's >90% purity by SDS-PAGE ensures the dominant band represents CSTL1, minimizing ambiguity in band assignment.

How should I store and handle Cystatin Like 1 (Recombinant) to maintain activity after arrival?

Store at -20°C immediately upon receipt. The protein is supplied in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol in single-use aliquots to eliminate repeated freeze-thaw cycles, which are the primary cause of activity loss in secreted cystatin-family proteins. Do not refreeze a thawed aliquot. For short-term bench use (same day), keep on ice; for use within 1–2 days, store the thawed aliquot at 4°C. Carrier protein (0.1% BSA) may be added if further dilutions below ~10 µg/mL are needed to reduce adsorptive losses on tube surfaces.