Gastricsin (Recombinant)

Frequently Asked Questions

What molecular weight band should I expect for recombinant Gastricsin on SDS-PAGE or Western blot?

Under denaturing SDS-PAGE conditions, recombinant Gastricsin runs at approximately 42–44 kDa, reflecting the mature processed form produced in HEK293 cells. The full-length zymogen precursor (pepsinogen C) is 388 amino acids with a predicted unglycosylated MW of ~42.8 kDa, but HEK293-derived glycosylation typically shifts the apparent MW slightly upward. If you observe a ~44–46 kDa band under your gel conditions, that is expected. The signal should collapse to the unglycosylated backbone (~42–43 kDa) after PNGase F treatment, which is a useful confirmation of correct identity.

Is the recombinant Gastricsin supplied as the zymogen (pepsinogen C) or the activated mature enzyme?

The product is supplied as the active mature enzyme form, not the full-length zymogen. Autocatalytic removal of the prosegment occurs under acidic conditions during processing; our HEK293-expressed construct is designed to yield the secreted, activated protease. This matters for activity assays: you do not need an acid-activation step before use. If your experiment specifically requires the inactive zymogen to study activation kinetics, note that this recombinant is not appropriate for that application without re-engineering the construct.

What substrates and pH conditions work best for a Gastricsin (EC 3.4.23.3) activity assay?

Gastricsin is most active in the pH 3.0–4.0 range, with a notably higher pH optimum than pepsin A — a key distinguishing feature. For fluorogenic activity assays, a FRET-based peptide substrate such as MoCAc-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Leu-Lys(Dnp)-D-Arg-NH2 or a hemoglobin cleavage assay at pH 3.5 in 50 mM sodium acetate buffer are established options in the literature. Avoid assay buffers above pH 5.5, as catalytic activity drops sharply. The storage buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol) keeps the enzyme stable but should be diluted into your acidic assay buffer immediately before use.

What starting concentration should I use for a Gastricsin enzymatic activity assay?

A reasonable starting point for fluorogenic or hemoglobin-digestion assays is 10–50 nM recombinant Gastricsin in your acidic assay buffer (pH 3.0–4.0, 50 mM sodium acetate). At 10 nM, you should see robust signal with most FRET peptide substrates at 100 µM substrate concentration. Titrate enzyme concentration to confirm linearity before comparing inhibitor IC50 values across experiments. Because the protein is active as supplied, avoid pre-warming the enzyme above room temperature during setup — brief incubation on ice prior to adding substrate helps minimize autolysis at acidic pH.

Can I use Gastricsin (Recombinant) as a positive control for Western blot with the matched rabbit polyclonal antibody?

Yes — this is one of the primary intended applications. The matched antibody, RP-Gastricsin (see /anti-gastricsin-rabbit-polyclonal-antibody), is raised from the same lab and has been validated for Western blot against this recombinant. Load 20–50 ng of recombinant Gastricsin per lane alongside your lysate samples. You should detect a clean band at ~42–44 kDa. This gives you a defined positive control that confirms antibody performance and allows you to identify the correct band in complex gastric mucosal or cancer cell line lysates where endogenous Gastricsin expression can vary widely.

How much recombinant Gastricsin should I load as a Western blot positive control, and at what antibody dilution?

Load 20–50 ng per lane for a clean, non-saturating signal. Using RP-Gastricsin at a primary antibody dilution of 1:1,000–1:2,000 (in 5% non-fat milk or BSA in TBST) typically yields a strong band at 42–44 kDa with short ECL exposure times (30–120 seconds). If background is high in your lysate lanes, reduce the recombinant control to 10 ng — the antibody is sensitive enough to detect this amount. Always run the recombinant in a lane adjacent to your experimental samples so band migration can be directly compared on the same gel.

How should I store and handle recombinant Gastricsin to maintain enzymatic activity long-term?

Store at -20°C in single-use aliquots to avoid repeated freeze-thaw cycles, which progressively denature the active enzyme. The supplied buffer — 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol — stabilizes the protein during frozen storage. On the bench, keep aliquots on ice and use within 2–4 hours of thawing. Do not store thawed protein at 4°C beyond 24–48 hours. Purity is >90–95% by SDS-PAGE and endotoxin is <0.1 EU/µg by LAL, so no additional cleanup steps are needed before most cell-based or biochemical experiments.

How does recombinant Gastricsin differ from pepsin A (PGA), and does that matter for my inhibitor profiling experiment?

Gastricsin (pepsinogen C / PGC) and pepsin A (PGA) are related but distinct aspartic proteases with different pH optima and substrate specificities. Gastricsin has a pH optimum around 3.0–4.0 and preferential cleavage at sites flanked by hydrophobic and aromatic residues, but it differs from pepsin A in its relative tolerance for certain P1/P1' combinations. For inhibitor profiling, this distinction is critical: many pepstatin A analogs inhibit both enzymes, but selectivity windows can emerge at pH 3.5 vs. pH 2.0 assay conditions. Running parallel assays with both proteases at equimolar concentrations is the cleanest way to establish selectivity.