Cystatin A (Recombinant)
- Expression system
- HEK293
- Cat. #
- REC-CystatinA
In stock
- SKU
- REC-CystatinA
Target Overview
Cystatin A (UniProt P01040; gene CSTA) is a 98-amino acid intracellular cysteine proteinase inhibitor belonging to the stefin/cystatin type-1 family. Also known as Stefin-A and Cystatin-AS, it lacks the signal peptide and disulfide bonds characteristic of secreted family members, and localises to the cytoplasm where it reversibly inhibits lysosomal cysteine proteases, including cathepsins B, H, and L. Its compact, single-domain architecture makes the full-length 98-residue sequence tractable for recombinant expression without truncation. This recombinant is produced in HEK293 cells, providing mammalian post-translational processing in a human cellular background — a consideration when using the protein as an antigen standard or in binding assays where folding fidelity matters. The full-length human sequence (residues 1–98) is expressed, consistent with the native cytoplasmic form. Researchers use this recombinant across several experimental contexts: as a positive control antigen for Western blot and IHC antibody validation (a matched Triple Point Biologics antibody, SKU RP-CystatinA, is available for pairing); as an inhibitor standard in fluorogenic cysteine protease activity assays using substrates such as Z-Phe-Arg-AMC; and as a calibration standard in ELISA-based quantification of CSTA in cell lysates or tissue extracts. It is also used as a binding partner in pull-down and co-immunoprecipitation experiments interrogating CSTA protein–protein interactions, including with integrins and structural components of desmosomes. The defined sequence and human expression system make it suitable for surface plasmon resonance (SPR) or biolayer interferometry (BLI) studies measuring inhibitor–protease binding kinetics.
Background
Applications
- Cysteine protease inhibition assay: dose-dependent inhibition of cathepsin B or L using fluorogenic substrates (e.g., Z-Phe-Arg-AMC) to determine Ki
- Antibody validation positive control: Western blot and IHC confirmation using matched Triple Point Biologics antibody SKU RP-CystatinA
- ELISA calibration standard: quantification of endogenous CSTA in keratinocyte or airway epithelial cell lysates
- Recombinant protein pull-down / co-IP: identification of CSTA-interacting partners including integrins and desmosomal proteins
- Cell stimulation assay: exogenous addition to glioblastoma or macrophage co-culture models to interrogate ITGB4–TGFB1 signalling
- SPR or BLI binding kinetics: measurement of CSTA association and dissociation rates with target cysteine proteases or candidate binding partners
- Ferroptosis research tool: use in cell-based assays testing CSTA overexpression or supplementation effects on ferroptotic endpoints in immune cell models
References
- Xie S et al. The SPRR2A-CSTA Axis Drives IL-17A-Induced Squamous Metaplasia and Steroid Resistance in Allergic Rhinitis. J Allergy Clin Immunol. 2026. doi:10.1016/j.jaci.2026.05.024. PMID: 42269941.
- Shen X et al. CSTA and DDX24: potential biomarkers regulating ferroptosis in sepsis and their diagnostic value. BMC Med Genomics. 2026. doi:10.1186/s12920-026-02358-x. PMID: 41952180.
- Wu J et al. M2-like GAMs secreting CSTA drive glioblastoma progression via the ITGB4-TGFB1 feedback axis. J Transl Med. 2026. doi:10.1186/s12967-026-08009-0. PMID: 41832564.
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 band should I expect for recombinant Cystatin A on SDS-PAGE or Western blot?
The full-length human Cystatin A sequence (residues 1–98) has a calculated molecular weight of approximately 11 kDa. On reducing SDS-PAGE, expect a single band migrating at roughly 11–12 kDa, confirmed at >90–95% purity by SDS-PAGE. Cystatin A lacks disulfide bonds, so reducing and non-reducing conditions should yield the same apparent band position. If you observe a slightly higher apparent MW under certain gel conditions, this is consistent with the known behavior of small, highly charged proteins on polyacrylamide gels.
Is this recombinant Cystatin A the full-length cytoplasmic isoform or a truncated fragment?
This recombinant represents the full-length human Cystatin A (UniProt P01040, residues 1–98), consistent with the native cytoplasmic stefin/type-1 cystatin form. It lacks a signal peptide — as expected for an intracellular cysteine protease inhibitor — and contains no disulfide bonds. No truncation or tag is introduced that would alter the native inhibitory domain architecture. This makes it suitable for functional inhibition assays as well as use as a structurally faithful antigen standard.
Which cysteine proteases does recombinant Cystatin A inhibit, and what substrates can I use to measure activity?
Cystatin A reversibly inhibits lysosomal cysteine proteases cathepsins B, H, and L. To measure inhibitory activity, pre-incubate recombinant Cystatin A with your target cathepsin (e.g., cathepsin B) before adding a fluorogenic substrate such as Z-Arg-Arg-AMC (for cathepsin B) or Z-Phe-Arg-AMC (for cathepsins B and L). Monitor AMC release at Ex/Em 360/460 nm. Reported Ki values for Cystatin A against cathepsin B are in the low nanomolar range; titrate your inhibitor from 1–500 nM to define your IC50 under your specific assay conditions.
What buffer conditions are recommended for Cystatin A recombinant protein activity assays?
The protein is supplied in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol — suitable for storage and most binding assays. For cathepsin inhibition assays, cathepsins B, H, and L are typically active at mildly acidic pH (5.0–6.5) in sodium acetate or MES buffer supplemented with 2–5 mM DTT and 1 mM EDTA to maintain the active-site thiol. Dilute recombinant Cystatin A into your assay buffer immediately before use. The glycerol in the storage buffer is compatible at the dilutions typically used (≥1:10) without interfering with fluorogenic readouts.
What starting concentration of recombinant Cystatin A should I use in a cathepsin inhibition assay?
A reasonable starting range is 10–100 nM recombinant Cystatin A when assaying against cathepsin B or L at 1–5 nM enzyme, giving you an inhibitor:enzyme ratio of 10:1 to 100:1 to observe near-complete inhibition. To determine IC50, run a 7–10 point dilution series from approximately 0.1 nM to 1 µM. Because Cystatin A is a tight-binding inhibitor (Ki in the low nM range for cathepsin B), use the Morrison equation for tight-binding inhibitor analysis rather than the standard Cheng-Prusoff approximation for accurate Ki estimation.
Can I use recombinant Cystatin A as a positive control antigen on a Western blot with the matched TPB antibody?
Yes — recombinant Cystatin A (REC-CystatinA) is designed to pair directly with the matched rabbit polyclonal antibody RP-CystatinA (/anti-cystatin-a-rabbit-polyclonal-antibody), both produced from the same human Cystatin A sequence in our lab. Load 10–50 ng of recombinant protein per lane on a 15–18% polyacrylamide gel to resolve the ~11 kDa band cleanly. Use this lane alongside your cell lysate samples to confirm antibody specificity and verify transfer efficiency of low-MW proteins, which can be problematic on standard PVDF membranes without methanol adjustment.
How much recombinant Cystatin A should I load for Western blot positive control, and will the band be detectable?
Load 10–50 ng per lane when using RP-CystatinA at its recommended dilution (refer to the antibody datasheet). At 25 ng, the ~11 kDa band is consistently detectable with a standard HRP-conjugated secondary antibody and ECL substrate in a 3–5 minute exposure. Because Cystatin A is a small protein, use a 15–18% SDS-PAGE gel or a Tris-tricine system, and transfer at low voltage (30 V overnight) onto 0.2 µm PVDF to minimize diffusion and loss of the small protein through the membrane.
How should I store and handle recombinant Cystatin A to maintain activity, and what is the shelf life?
Store at –20°C in single-use aliquots immediately upon receipt. The 10% glycerol in the storage buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol) provides cryoprotection. Avoid repeated freeze-thaw cycles, as even two additional cycles can measurably reduce inhibitory activity for small, soluble inhibitor proteins. When properly stored, the protein is stable for at least 12 months. After thawing, keep on ice and use within the same working session. Do not dilute working stocks in carrier-free PBS unless you add 0.1% BSA to prevent adsorption losses at low concentrations (<10 nM).
Validation imagery coming soon
Western blot validation figures for REC-CystatinA 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.