Prolyl Oligopeptidase (Recombinant)

Frequently Asked Questions

What is the expected molecular weight of recombinant Prolyl Oligopeptidase on SDS-PAGE or Western blot?

Recombinant human Prolyl Oligopeptidase (PREP, P48147) is a full-length 710-amino acid protein with a predicted molecular weight of approximately 80 kDa. On reducing SDS-PAGE, the primary band typically migrates at ~80–82 kDa. No signal peptide cleavage or propeptide processing is known for this cytoplasmic serine protease, so you should not see a processed lower-molecular-weight form under standard denaturing conditions. Purity is >90% by SDS-PAGE. When running as a Western blot positive control alongside our matched antibody RP-Prolyl Oligopeptidase, load alongside your lysate lane for direct band comparison.

Is recombinant Prolyl Oligopeptidase a processed or single-chain form, and are there known isoforms?

Human PREP is translated as a single 710-residue polypeptide with no known signal peptide or propeptide that requires proteolytic removal for activation. The enzyme is catalytically active as a monomer in its full-length form. UniProt P48147 lists no confirmed alternatively spliced isoforms that alter the catalytic domain. This recombinant covers the complete 710-residue sequence expressed in HEK293 cells, meaning the protein retains the native α/β-hydrolase fold and the β-propeller domain that gates substrate access. You should expect a single predominant band at ~80 kDa with no evidence of a two-chain or zymogen species.

What peptide substrates does Prolyl Oligopeptidase cleave, and which fluorogenic substrate works best in vitro?

PREP (EC 3.4.21.26) hydrolyses the peptide bond on the C-terminal side of prolyl residues in peptides up to ~30 amino acids. For fluorogenic activity assays, Z-Gly-Pro-AMC and Suc-Gly-Pro-MCA are the most widely used substrates and are well-suited to plate-reader formats (Ex/Em ~360/460 nm for AMC; ~380/460 nm for MCA). Typical substrate concentrations range from 50–500 µM depending on your Km determinations. Note that PREP does not cleave proteins or large polypeptides — the β-propeller domain sterically excludes substrates longer than ~30 residues, which is an important selectivity consideration when designing peptide cleavage panels.

What assay buffer conditions are recommended for Prolyl Oligopeptidase activity assays?

The protein is supplied in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol and is active in this storage buffer. For fluorogenic substrate assays, a common working buffer is 50 mM Tris-HCl or sodium phosphate pH 7.0–7.5, with 1–2 mM DTT or TCEP to maintain the free cysteine residues that contribute to stability. Avoid PMSF and other serine protease inhibitors, which will inhibit PREP directly. Metal chelators (EDTA, EGTA) are generally tolerated since PREP is not a metalloprotease. Keep glycerol ≤2% in the final assay volume to minimize effects on fluorescence readings.

What starting concentration of recombinant Prolyl Oligopeptidase should I use in an IC50 inhibitor assay?

For competitive inhibition assays using Z-Gly-Pro-AMC, a starting enzyme concentration of 1–5 nM recombinant PREP typically gives a linear signal response with 100–200 µM substrate over a 30–60 minute endpoint read. Titrate enzyme first to establish a concentration that produces a RFU/min within your detector's linear range before introducing inhibitors. Because PREP activity is sensitive to compound aggregation artifacts at high inhibitor concentrations, include Triton X-100 at 0.01% as a counter-screen control. Protein concentration of the supplied stock should be confirmed by BCA or A280 before dilution to ensure accurate enzyme:inhibitor molar ratios.

Can I use recombinant Prolyl Oligopeptidase as a positive control for Western blot with the RP-Prolyl Oligopeptidase antibody?

Yes — this is a primary intended use case. The matched rabbit polyclonal antibody RP-Prolyl Oligopeptidase (/anti-prolyl-oligopeptidase-rabbit-polyclonal-antibody) was raised and validated in the same TPB laboratory, and the two products are guaranteed compatible for Western blot positive control applications. Load 10–50 ng of recombinant PREP per lane on a standard 8–10% SDS-PAGE gel; this yields a clean ~80 kDa band at typical antibody dilutions of 1:500–1:2,000. Running recombinant alongside cell lysate lets you confirm band identity, assess antibody specificity, and establish a reliable reference when optimizing lysis conditions or new tissue panels.

How much recombinant Prolyl Oligopeptidase should I load for Western blot antibody validation experiments?

For antibody validation with RP-Prolyl Oligopeptidase, 20–50 ng per lane is a practical starting point when using standard ECL detection. If you are running a dilution series to determine the linear detection range of the antibody, loading 5, 10, 25, and 50 ng in adjacent lanes is informative. Avoid loading >100 ng, as band broadening and potential secondary bands from minor contaminants may complicate interpretation. Because the recombinant is >90% pure by SDS-PAGE, any bands other than ~80 kDa should be treated as assay artifacts rather than PREP isoforms or processing products.

How should I store and handle recombinant Prolyl Oligopeptidase to preserve catalytic activity over time?

The protein is shipped and stored at -20°C in single-use aliquots in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol. Avoid repeated freeze-thaw cycles — each cycle can reduce specific activity measurably for serine proteases. Upon first use, thaw on ice, briefly centrifuge at 10,000 × g to pellet any microaggregates, and dilute directly into assay buffer. Working dilutions in Tris-HCl pH 7.4–7.5 with 0.1% BSA as carrier are stable for 4–8 hours on ice. For longer-term bench stability, supplementing diluted aliquots with 0.1% BSA and 1 mM DTT is advisable. Endotoxin is <0.1 EU/µg by LAL assay.