Cystatin B (Recombinant)

Frequently Asked Questions

What molecular weight should I expect for recombinant Cystatin B on SDS-PAGE and Western blot?

The full-length human Cystatin B sequence is 98 amino acids, giving a predicted molecular weight of approximately 11 kDa. On SDS-PAGE under reducing conditions, the HEK293-expressed recombinant typically migrates at 11–13 kDa; slight upward shifts relative to the predicted MW are common and reflect mammalian post-translational modifications such as N-terminal acetylation. Purity is >95% by SDS-PAGE. When running Western blot positive controls alongside our matched antibody RP-CystatinB, load 20–50 ng per lane to get a clean, well-resolved band in that 11–13 kDa window.

Which isoform of Cystatin B does this recombinant represent, and is it the full-length sequence?

This recombinant corresponds to the canonical full-length human Cystatin B isoform 1 (UniProt P04080-1), encoding all 98 residues. No signal peptide cleavage occurs because Cystatin B is a type-1 stefin and is naturally cytoplasmic — the expressed product therefore retains the complete N-terminus. There are no disulfide bonds to worry about under standard reducing electrophoresis conditions. If your experiment targets the progressive myoclonus epilepsy-associated variants, note that those single-residue substitutions are not present in this wild-type construct.

Which cathepsins does recombinant Cystatin B inhibit, and what fluorogenic substrates should I use to measure inhibition?

Cystatin B is a tight-binding, reversible inhibitor of cysteine cathepsins B, H, and L. For activity inhibition assays, use cathepsin B with Z-Arg-Arg-AMC (excitation 380 nm / emission 460 nm), cathepsin L with Z-Phe-Arg-AMC, and cathepsin H with H-Arg-AMC. Run reactions in 100 mM sodium acetate pH 5.5, 1 mM EDTA, 2 mM DTT at 37°C — the reducing agent is critical to keep the active-site cysteine of the cathepsin reduced. Pre-incubate Cystatin B with cathepsin for 10 minutes before adding substrate to allow tight-binding equilibrium.

What is a good starting concentration of recombinant Cystatin B for a cathepsin inhibition IC50 assay?

Published Ki values for Cystatin B against cathepsins B, H, and L are in the low-to-sub nanomolar range (0.3–7 nM depending on the cathepsin). For IC50 determination, start with a cathepsin concentration near or below your Cystatin B Ki (e.g., 1–5 nM cathepsin) and titrate Cystatin B across 0.01–100 nM. Because this is a tight-binding inhibitor, standard Cheng-Prusoff corrections may underestimate potency; consider using Morrison's tight-binding IC50 equation for accurate Ki extraction. The HEK293 expression system avoids refolding artifacts that can artificially inflate IC50 values seen with bacterially expressed material.

What buffer is recombinant Cystatin B supplied in, and how should I dilute it for functional assays?

The protein is supplied in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol. This storage buffer is compatible with most downstream assay formats, but because cathepsin activity assays run at pH 5.5, prepare your working dilutions in the assay buffer immediately before use rather than pre-diluting in Tris pH 7.5. For Western blot loading controls, dilute directly into 1× Laemmli sample buffer. Avoid repeated freeze-thaw — aliquot upon receipt and store at -20°C. Properly stored aliquots are stable for at least 12 months without significant loss of inhibitory activity.

Can I use recombinant Cystatin B as a positive control for Western blot with the RP-CystatinB antibody?

Yes — this recombinant is the ideal positive control for RP-CystatinB (/anti-cystatin-b-rabbit-polyclonal-antibody). Load 20–50 ng of recombinant Cystatin B alongside your cell lysate samples; the antibody should detect a clean band at 11–13 kDa under standard reducing SDS-PAGE conditions. Because the recombinant is expressed from the same full-length sequence used to validate RP-CystatinB, you get a defined, concentration-calibrated positive control rather than relying on endogenous expression levels in your cell line. This pairing is particularly useful when validating RP-CystatinB in a new tissue type or species-matched lysate.

How much recombinant Cystatin B should I load for a Western blot positive control lane?

20–50 ng per lane is a reliable starting point when using RP-CystatinB at a 1:1,000–1:2,000 dilution with standard chemiluminescent detection. At 20 ng you should resolve a clear, specific band at 11–13 kDa with minimal background. If you are using a more sensitive detection system (e.g., fluorescent secondary antibody with quantitative imaging), 5–10 ng may suffice. Run a small titration (5, 20, 50 ng) the first time you establish the assay so you can choose a load that sits within the linear dynamic range of your detection system and matches endogenous signal intensity in your lysate lanes.

Why use HEK293-expressed recombinant Cystatin B instead of E. coli-expressed material for cathepsin inhibition assays?

Cystatin B expressed in E. coli frequently requires denaturation and refolding steps that can leave a portion of the protein in non-native conformations with reduced cathepsin-binding competence. This lowers the apparent inhibitory potency and introduces lot-to-lot variability in Ki measurements. The HEK293 system provides mammalian post-translational processing — including N-terminal acetylation — and produces natively folded material directly in solution. The result is more reproducible inhibition kinetics, particularly important when comparing IC50 values across cathepsin isoforms or screening Cystatin B against inhibitor-competition assays.