Cystatin C (Recombinant)
- Expression system
- HEK293
- Cat. #
- REC-CystatinC
In stock
- SKU
- REC-CystatinC
Target Overview
Cystatin C (gene: CST3; UniProt P01034) is a 146-amino-acid secreted cysteine protease inhibitor belonging to the type 2 cystatin superfamily. It is expressed ubiquitously and constitutively secreted into virtually all human body fluids, where it functions as a tightly binding, reversible inhibitor of cysteine endopeptidases — principally cathepsins B, H, L, and S — through a wedge mechanism involving both N-terminal and loop contact regions. This recombinant is produced in HEK293 mammalian cells, providing post-translational processing consistent with the native secreted form. Mammalian expression is preferred for Cystatin C over prokaryotic systems because the native protein requires correct disulfide bond formation (Cys73–Cys83) for full inhibitory activity; misfolded bacterially derived material can aggregate or display reduced Ki values against target cathepsins. The HEK293-expressed material is therefore well suited where authentic inhibitory activity is a prerequisite. Researchers use this recombinant in several complementary workflows. In enzymatic inhibition assays, it serves as a reference inhibitor alongside synthetic cysteine protease substrates (e.g., Z-Phe-Arg-AMC for cathepsin B/L) to establish Ki or IC50 benchmarks. In inhibitor screening campaigns, it is used as a competitor standard to evaluate selectivity of small-molecule candidates. For antibody validation workflows, the full-length recombinant provides a well-characterised positive control in Western blot and dot-blot experiments; researchers using this protein for that purpose can pair it directly with the matched Triple Point Biologics antibody (SKU: RP-CystatinC), which has been validated for Western blot against human Cystatin C. The protein also serves as a calibration standard in immunoassay development and as an antigen for surface plasmon resonance (SPR) or biolayer interferometry (BLI) binding studies.
Background
Applications
- Cysteine protease inhibition assay: measurement of Ki against cathepsin B or L using fluorogenic substrate Z-Phe-Arg-AMC
- Inhibitor selectivity screening: use as competitor reference standard to rank small-molecule cysteine protease inhibitor candidates
- Antibody validation positive control: Western blot and dot-blot confirmation of anti-Cystatin C antibody specificity, paired with SKU RP-CystatinC
- Immunoassay calibration: serial dilution standard curve preparation for sandwich ELISA quantification of Cystatin C in biological fluids
- Surface plasmon resonance (SPR) or biolayer interferometry (BLI): analyte or ligand for measuring binding kinetics of anti-Cystatin C antibodies or cathepsin binding partners
- Cell-based extracellular matrix degradation assay: exogenous addition to modulate cathepsin-mediated matrix remodelling in invasion or degradation models
- Recombinant protein stability and aggregation study: reference material for investigating domain-swapped dimer formation under defined buffer and pH conditions
References
Note: The PubMed citations provided with this product record do not directly describe Cystatin C research and are therefore not cited in the background text above. No references are listed to avoid misattribution. Only literature directly relevant to this recombinant protein is cited in Triple Point Biologics reference pages.Additional Specifications
| Storage Buffer | 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol |
|---|---|
| Endotoxin Level | <0.1 EU/µg by LAL |
| Purity (%) | >90% by SDS-PAGE |
| Expression System | HEK293 |
| Subcellular Localization | Secreted; extracellular fluids |
Frequently Asked Questions
What molecular weight does recombinant Cystatin C run at on SDS-PAGE and Western blot?
The mature secreted form of Cystatin C (residues 27–146 after signal peptide cleavage) has a calculated MW of approximately 13.3 kDa. On reducing SDS-PAGE, the HEK293-expressed recombinant typically migrates at 13–15 kDa; minor upward shifts relative to the calculated mass are common due to glycosylation heterogeneity at Asn61. Under non-reducing conditions, the intramolecular Cys73–Cys83 disulfide is preserved, so mobility is essentially unchanged. Expect a single predominant band at >90% purity when run alongside a broad-range MW ladder.
Which isoform or processing form of Cystatin C does the recombinant represent — full-length or mature?
This recombinant represents the mature, secreted form of human Cystatin C (UniProt P01034, residues 27–146), with the 26-amino-acid signal peptide removed co-translationally in the HEK293 expression system. No further proteolytic processing is known to occur in normal physiology, so the 120-residue mature chain is the functionally relevant species found in plasma, CSF, and urine. The N-terminal region (Gly28 onwards) and both loop regions (L1 and L2) remain intact and are required for full wedge-mechanism inhibition of cathepsins B, H, L, and S.
What does recombinant Cystatin C inhibit and what are the target cathepsins?
Cystatin C is a tight-binding, reversible inhibitor of cysteine endopeptidases. Its primary targets are lysosomal cathepsins B, H, L, and S, with reported Ki values in the low picomolar to low nanomolar range (e.g., Ki ~0.008 nM for cathepsin L, ~0.25 nM for cathepsin B under assay-dependent conditions). Inhibition proceeds via a wedge mechanism: the N-terminal region and two hairpin loops (L1: Gln55–Gly59; L2: Pro105–Trp106) contact the active-site cleft of the target protease. Correctly folded, disulfide-intact material from mammalian expression is required to achieve these Ki values; bacterially expressed, misfolded material shows significantly reduced potency.
What substrate and assay conditions should I use to measure Cystatin C inhibitory activity against cathepsin B?
A standard fluorogenic assay uses Z-Arg-Arg-AMC (25–100 µM) as substrate with human or bovine cathepsin B in 50 mM sodium acetate, 1 mM EDTA, 1 mM DTT, pH 5.5 — conditions that fully activate the protease. Pre-incubate recombinant Cystatin C (serial dilutions from 0.1 nM to 10 nM) with cathepsin B for 10–15 min at 37°C before adding substrate. Measure AMC release at Ex 380 nm / Em 460 nm. Run a no-inhibitor control to define 100% activity. Expect IC50 in the low nanomolar range; exact values depend on cathepsin B concentration relative to the tight-binding threshold.
What starting concentration of recombinant Cystatin C is recommended for a cathepsin inhibition dose-response experiment?
For a dose-response inhibition curve against cathepsin L or B, start with a top concentration of 100 nM recombinant Cystatin C and titrate down 10-fold to ~0.01 nM (7–8 point curve). Keep cathepsin concentration at or below your anticipated Ki to avoid the tight-binding artifact that compresses the apparent IC50. The recombinant is supplied at a defined concentration in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol; dilute into your assay buffer immediately before use. Avoid carrying >0.5% glycerol into fluorogenic assays, as it can interfere with AMC fluorescence at high concentrations.
Can I use this recombinant Cystatin C as a positive control for Western blot with TPB antibody RP-CystatinC?
Yes — this is one of the primary use cases for pairing REC-CystatinC with RP-CystatinC. Load 5–20 ng of recombinant per lane on a 15% or 4–20% gradient SDS-PAGE gel under reducing conditions; the antibody will detect the ~13–15 kDa band. RP-CystatinC is a rabbit polyclonal raised against human Cystatin C and is validated for Western blot. Using the recombinant as a positive control allows you to confirm antibody sensitivity, set exposure, and verify that any bands in your sample lysate at ~13–15 kDa are genuine Cystatin C signal rather than non-specific background.
How much recombinant Cystatin C should I load for Western blot positive control, and what band should I expect?
Load 10–20 ng per lane as a starting point alongside your cell or tissue lysates. At this amount, RP-CystatinC (used at 1:1,000–1:2,000 dilution with standard HRP-conjugated secondary) reliably detects a band at approximately 13–15 kDa, slightly above the calculated 13.3 kDa due to partial N-glycosylation. If running on a 12% or 15% gel, ensure your running conditions resolve this low-MW range adequately — run until the 10 kDa marker is near the gel front. The recombinant gives a clean single band useful for gauging antibody specificity against endogenous Cystatin C in secreted fractions or conditioned media.
How should I store and handle recombinant Cystatin C to preserve inhibitory activity, and what is the shelf life?
The protein is supplied in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol and should be stored at −20°C in single-use aliquots. Repeated freeze-thaw cycles degrade the Cys73–Cys83 disulfide bond critical for full inhibitory activity; plan aliquot sizes to match single-experiment needs. Shelf life is 12 months from receipt at −20°C. Upon thaw, keep on ice and use within the same working day. Do not dilute stock solutions far in advance — diluted protein at sub-nanomolar concentrations is prone to adsorption losses on plastic surfaces; consider adding 0.01% BSA to dilution buffers for very low-concentration working solutions.
Validation imagery coming soon
Western blot validation figures for REC-CystatinC will be published here as they are produced in-house.
If you would like to see existing validation data for this antibody before publication, request a sample copy.