Cystatin-9 (Recombinant)
- Expression system
- HEK293
- Cat. #
- REC-Cystatin9
In stock
- SKU
- REC-Cystatin9
Target Overview
Cystatin-9 (CST9; UniProt Q5W186) is a secreted, 159-amino-acid member of the cystatin superfamily of cysteine protease inhibitors. This recombinant form is produced in HEK293 cells, an expression system that supports the post-translational processing and secretory pathway handling relevant for a naturally secreted protein. The full-length 159-residue sequence spans the complete mature polypeptide as annotated in UniProt. As a secreted cystatin-family protein, CST9 is positioned to act extracellularly, where cystatins typically modulate the activity of cysteine cathepsins and related proteases. This makes the recombinant protein directly applicable to in vitro cysteine protease inhibition assays, where it can be titrated against target cathepsins to establish inhibitory kinetics or confirm substrate competition. Beyond classic protease inhibition, CST9 has been characterised for immunomodulatory and antimicrobial functions — notably against the intracellular Gram-negative pathogen Francisella tularensis — and has been studied in the context of hematopoietic differentiation and inflammation. This functional profile makes the recombinant protein a useful tool antigen for researchers studying innate immune modulation, host–pathogen interaction mechanisms, and myeloid cell biology. Researchers requiring a well-defined positive control or capture antigen for Western blot or immunohistochemistry will find this reagent directly compatible with the matched Triple Point Biologics anti-Cystatin-9 antibody (SKU: RP-Cystatin9), which has been validated against human targets. The recombinant protein serves both as an antibody validation standard and as a functional tool in activity and binding assays. Its HEK293 expression ensures mammalian glycosylation context where relevant to binding or immune recognition studies.
Background
Applications
- Cysteine protease inhibition assay — titration of recombinant CST9 against cathepsin B, H, L, or S to determine Ki or IC50 values
- Antibody validation positive control — use as defined antigen to confirm band identity of anti-Cystatin-9 (RP-Cystatin9) in Western blot
- IHC signal specificity control — spike-in or pre-absorption control to confirm anti-CST9 staining specificity in tissue sections
- Antimicrobial function studies — in vitro assays examining CST9 activity against Francisella tularensis or other Gram-negative bacterial challenge models
- Immunomodulation assays — stimulation of primary hematopoietic or myeloid cell cultures to study CST9 effects on differentiation or inflammatory cytokine output
- ELISA standard or capture antigen — use as calibration standard or solid-phase capture protein in sandwich ELISA development for CST9 detection in biological fluids
- Protein–protein interaction binding assay — surface plasmon resonance or biolayer interferometry to characterise CST9 binding to target proteases or immune receptors
- Recombinant antigen for immunisation — use as defined immunogen in production or benchmarking of research-grade anti-CST9 antibodies
References
- Saldarriaga Cartagena AM et al. Toxoplasma cyst wall CST9 elicits an acute-associated humoral response in humans and mice and protects against chronic infection in immunized mice. Microb Pathog. 2025. doi:10.1016/j.micpath.2025.107638 PMID: 40287105
- Boukhalfa W et al. Decoding the genetic relationship between Alzheimer's disease and type 2 diabetes: potential risk variants and future direction for North Africa. Front Aging Neurosci. 2023. doi:10.3389/fnagi.2023.1114810 PMID: 37342358
- Li XH et al. cfDNA Methylation Profiles and T-Cell Differentiation in Women with Endometrial Polyps. Cells. 2022. doi:10.3390/cells11243989 PMID: 36552753
- Mohamed SA et al. GWAS in people of Middle Eastern descent reveals a locus protective of kidney function-a cross-sectional study. BMC Med. 2022. doi:10.1186/s12916-022-02267-7 PMID: 35227251
- Yang M et al. Kininogen-1 as a protein biomarker for schizophrenia through mass spectrometry and genetic association analyses. PeerJ. 2019. doi:10.7717/peerj.7327 PMID: 31346501
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 should I expect for recombinant Cystatin-9 on SDS-PAGE or Western blot?
The full-length 159-residue CST9 polypeptide has a calculated molecular weight of approximately 17–18 kDa. On reducing SDS-PAGE, HEK293-expressed protein may run slightly higher — typically 19–22 kDa — due to glycosylation or other post-translational modifications acquired through the secretory pathway. Purity is >90% by SDS-PAGE. If you are running a Western blot positive control alongside our matched antibody RP-Cystatin9, expect the predominant band in that 19–22 kDa window under standard reducing conditions.
Is recombinant Cystatin-9 the full-length mature protein or a truncated fragment?
This recombinant spans the complete 159-amino-acid sequence of the mature CST9 polypeptide as annotated in UniProt Q5W186, expressed in HEK293 cells with full secretory-pathway processing. It is not a truncated fragment or bacterial refolding product. Because the protein traverses the HEK293 secretory pathway, signal-peptide cleavage and any associated modifications mirror the endogenous processing context more closely than E. coli-derived material would.
Which cysteine proteases does Cystatin-9 inhibit, and what substrates work for an in vitro inhibition assay?
CST9 belongs to the cystatin superfamily and is expected to inhibit papain-family cysteine cathepsins (cathepsins B, L, S, and related enzymes). For a standard in vitro inhibition assay, fluorogenic substrates such as Z-Phe-Arg-AMC (for cathepsin B/L) or Z-Leu-Arg-AMC work well. Pre-incubate CST9 with the target cathepsin in assay buffer at 37°C for 10–15 minutes before adding substrate. This allows equilibrium inhibitor binding and produces clean dose-response curves for IC50 determination.
What buffer conditions are recommended for Cystatin-9 activity or inhibition assays?
The protein is supplied in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol. For cathepsin inhibition assays, dilute into a standard assay buffer — commonly 50–100 mM sodium acetate or sodium phosphate, pH 5.5–6.5, supplemented with 2–5 mM DTT or EDTA to maintain reducing conditions and cathepsin activity. The glycerol in the storage buffer is compatible at typical working dilutions (≥1:10) and will not interfere with fluorescence readouts. Avoid prolonged exposure to non-reducing, alkaline conditions, which can affect cystatin activity.
What starting concentration of recombinant Cystatin-9 should I use to determine IC50 against cathepsin B?
A reasonable starting range is 1–500 nM CST9, titrated in 3- to 5-fold serial dilutions against a fixed cathepsin B concentration (typically 1–5 nM enzyme). Published IC50 values for cystatin-family proteins against cathepsin B often fall in the low-nanomolar range (1–50 nM), so design your series to bracket that window. Confirm enzyme concentration by active-site titration with E-64 before the assay to ensure stoichiometry-based comparisons are meaningful.
Can I use recombinant Cystatin-9 as a Western blot positive control with the RP-Cystatin9 rabbit polyclonal antibody?
Yes — this is a primary intended use case. The recombinant protein (REC-Cystatin9) and the matched rabbit polyclonal antibody (RP-Cystatin9) are derived and validated in the same laboratory, guaranteeing epitope compatibility. Load 50–200 ng of recombinant protein per lane alongside your cell lysate samples. The antibody has been validated for Western blot, so you can expect a clean band at 19–22 kDa. This pairing is particularly useful when establishing antibody working dilutions or confirming antibody specificity in a new cell or tissue context.
How much recombinant Cystatin-9 should I load as a positive control for Western blot, and at what antibody dilution?
Load 50–200 ng of REC-Cystatin9 per lane for a robust positive control signal. Start with RP-Cystatin9 at a 1:1,000–1:2,000 dilution in 5% non-fat milk or BSA/TBST, then adjust based on your detection system sensitivity. HRP-conjugated secondary at 1:5,000–1:10,000 typically gives a clean band at 19–22 kDa with minimal background. If signal is too strong at 50 ng, stepping down to 10–25 ng is sufficient for most chemiluminescent systems. See the RP-Cystatin9 product page at /anti-cystatin-9-rabbit-polyclonal-antibody for validated dilution data.
How should I store and handle recombinant Cystatin-9 to preserve 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) acts as a cryoprotectant. Avoid repeated freeze-thaw cycles — each cycle can measurably reduce inhibitory activity. Working aliquots can be held at 4°C for up to 48 hours without significant loss. Endotoxin is <0.1 EU/µg by LAL, making the protein suitable for cell-based assays where endotoxin interference is a concern. Under correct storage, shelf life is 12 months from the date of receipt.
Validation imagery coming soon
Western blot validation figures for REC-Cystatin9 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.