Cystatin-8 (Recombinant)

Frequently Asked Questions

What is the expected molecular weight of recombinant Cystatin-8 on SDS-PAGE and Western blot?

Recombinant Cystatin-8 (REC-Cystatin8) produced in HEK293 cells migrates at approximately 14–16 kDa on reducing SDS-PAGE, consistent with the mature secreted form of the CST8-encoded protein. HEK293 expression introduces eukaryotic glycosylation and other post-translational modifications, so the observed band may run slightly higher than the theoretical mass of ~11.5 kDa predicted from amino acid sequence alone. Purity is confirmed at >90–95% by Coomassie-stained SDS-PAGE before release. When used as a Western blot positive control alongside RP-Cystatin8, expect a clean single band in the 14–17 kDa window under reducing conditions.

Is recombinant Cystatin-8 the full-length protein or a processed isoform, and does it form amyloid in solution?

REC-Cystatin8 corresponds to the full-length mature secreted form of human CST8 (UniProt Q60676), expressed without a signal peptide but otherwise untruncated. As a CRES subgroup cystatin, Cystatin-8 is competent to form functional amyloid structures under appropriate conditions — behaviour documented for the native epididymal protein. The HEK293 expression system was specifically chosen to preserve native-like conformation critical for amyloid nucleation and cross-seeding studies. To minimize spontaneous fibrillation during storage, aliquots are maintained in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol at -20°C. Avoid extended incubation at 37°C in low-salt conditions if monomeric protein is required for your assay.

Does Cystatin-8 inhibit cysteine proteases like cathepsin B or L the same way classical cystatins do?

Cystatin-8 is a CRES subgroup member and diverges functionally from classical cystatin-2 subfamily inhibitors. Available literature indicates that CRES proteins, including Cystatin-8, exhibit attenuated or absent cysteine protease inhibitory activity toward canonical substrates such as cathepsin B and cathepsin L, in contrast to Cystatin-C. The predominant physiological role of Cystatin-8 appears to be structural — participating in amyloid matrix formation in the epididymal lumen — rather than direct protease inhibition. Researchers designing cysteine protease inhibition assays should treat REC-Cystatin8 accordingly and not assume IC50 values comparable to classical cystatins without empirical confirmation in their specific assay system.

What assay format and starting concentration are recommended for studying Cystatin-8 amyloid aggregation kinetics?

For thioflavin T (ThT) fluorescence-based aggregation kinetics, a starting concentration of 5–20 µM REC-Cystatin8 in a low-salt buffer (e.g., 20 mM sodium phosphate, pH 7.0) at 37°C with continuous or intermittent agitation is a practical starting point, consistent with published CRES-subgroup amyloid protocols. The standard storage buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol) should be diluted or exchanged prior to the assay, as high glycerol concentrations can suppress nucleation kinetics. For cross-seeding experiments, pre-formed seeds at 1–5% (v/v) of total protein are a reasonable titration range. Protein concentration should be verified by A280 after buffer exchange.

What buffer conditions are optimal for Cystatin-8 functional studies and does the storage buffer need to be exchanged?

The storage buffer — 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol — is designed for protein stability and long-term storage, not necessarily optimized for every downstream assay. For amyloid assembly or biophysical characterization (DLS, CD, AFM), dialysis or spin-column buffer exchange into a defined, glycerol-free buffer is recommended. For binding or co-immunoprecipitation studies, the storage buffer is generally compatible without exchange. Note that Cystatin-8 does not require Ca²⁺ supplementation — the 5 mM CaCl₂ additive in TPB metalloproteinase recombinants is not included in this formulation.

How much recombinant Cystatin-8 should I load as a Western blot positive control when using the RP-Cystatin8 antibody?

For Western blot positive control applications paired with RP-Cystatin8 (/anti-cystatin-8-rabbit-polyclonal-antibody), load 50–200 ng of REC-Cystatin8 per lane on a 15–18% Tris-glycine or 4–20% gradient gel. At 50 ng, a clean band at approximately 14–17 kDa is typically detectable with standard ECL detection and RP-Cystatin8 at a 1:1,000–1:2,000 dilution. Because REC-Cystatin8 and RP-Cystatin8 originate from the same TPB production series, compatibility is guaranteed — no need to empirically validate the antibody against a new recombinant batch from a different vendor.

Can I use recombinant Cystatin-8 to validate the RP-Cystatin8 rabbit polyclonal antibody for IHC on epididymal tissue sections?

REC-Cystatin8 is most directly useful for antibody validation in Western blot and dot-blot formats; for IHC validation on epididymal tissue, the recombinant can serve as a peptide-competition control by pre-incubating RP-Cystatin8 (see /anti-cystatin-8-rabbit-polyclonal-antibody) with excess antigen (typically 5–10 µg/mL recombinant protein) prior to tissue application. Abrogation of staining in competition confirms specificity. TPB's RP-Cystatin8 is validated for Western blot, and the matched recombinant provides a defined positive-control antigen for both modalities — a workflow TPB has supported for protease and inhibitor targets since 1994.

How should I handle, aliquot, and store recombinant Cystatin-8 to maintain stability and avoid freeze-thaw degradation?

REC-Cystatin8 is supplied in single-use aliquots in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol and should be stored at -20°C immediately upon receipt. Repeated freeze-thaw cycles accelerate aggregation — particularly relevant for a protein with intrinsic amyloidogenic character — so avoid refreezing thawed material. Upon thaw, briefly centrifuge at 10,000 × g for 2 minutes to pellet any microfibrillar aggregates before use. Working dilutions in assay-compatible buffers should be prepared fresh and kept on ice. Shelf life under recommended storage conditions is 12 months from the date of manufacture when freeze-thaw cycles are minimized.