Granzyme B (Recombinant)

Frequently Asked Questions

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

The full-length human Granzyme B sequence (UniProt P10144) predicts ~28 kDa, but the glycosylated, HEK293-expressed recombinant typically migrates at approximately 32–35 kDa under reducing SDS-PAGE conditions due to N-linked glycosylation. Under non-reducing conditions, the apparent MW may shift slightly. If your observed band falls outside this range, confirm that your gel percentage is appropriate (12–15% recommended) and that the supplied processed form matches the form your antibody was raised against. Check the lot-specific CoA for the exact observed band position.

Is the recombinant Granzyme B supplied as the full-length precursor or the processed active mature form?

The recombinant is produced in HEK293 cells, which support the signal-peptide cleavage and propeptide processing that yields the mature catalytic chain. The supplied protein represents the processed, active form rather than the zymogen precursor. Because the defined N-terminus matters for activity assays benchmarked against literature values, review the lot-specific CoA to confirm the exact processed residues included. Researchers designing N-terminus-sensitive experiments — such as those using substrates requiring a free catalytic serine environment — should take note of any His- or tag sequences that may be present.

What substrate cleavage specificity does recombinant Granzyme B have and which fluorogenic substrates work best for activity assays?

Granzyme B (EC 3.4.21.79) cleaves C-terminal to aspartate residues, the same primary specificity as the caspase family. The most widely validated fluorogenic substrates for activity confirmation are Ac-DEVD-AMC and IETD-AMC. In a standard kinetic assay, use 50–200 µM substrate in 50 mM HEPES pH 7.4, 150 mM NaCl, 0.1% CHAPS, at 37 °C, monitoring AMC release at Ex/Em 380/460 nm. Specific activity values can then be compared against the lot-specific CoA to verify that the preparation meets the stated activity specification before proceeding to inhibitor IC50 measurements.

What buffer conditions are optimal for Granzyme B activity assays and are there any additives to avoid?

For kinetic activity assays, 50 mM HEPES pH 7.4, 150 mM NaCl, 0.1% CHAPS at 37 °C is a well-established starting point. The storage buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol) is compatible with most assay formats when diluted at least 1:10 into assay buffer, reducing glycerol to ≤1%. Avoid divalent metal chelators such as EDTA at concentrations above 1 mM, which can affect the serine protease active-site environment indirectly. DTT at ≤1 mM is generally tolerated. Pre-warm enzyme on ice and equilibrate to assay temperature for 5 minutes before substrate addition.

What starting enzyme concentration should I use for Granzyme B IC50 inhibitor assays?

A typical starting point is 1–10 nM recombinant Granzyme B in the assay well, which provides sufficient AMC signal above background while remaining in a linear range compatible with the Morrison tight-binding equation if needed for potent inhibitors (Ki < 10× [E]). Confirm the active-site concentration by titration with a known irreversible inhibitor such as PMSF or a validated chloromethyl ketone before committing inhibitor stock to full IC50 curves. The lot CoA reports units/mg; calculate the nanomolar active-enzyme concentration from the specific activity to ensure assay conditions are appropriate.

Can I use recombinant Granzyme B as a positive control for Western blot with the matched anti-Granzyme B antibody RP-GranzymeB?

Yes — this is one of the most direct applications. Load 10–50 ng of recombinant Granzyme B per lane on a 12% SDS-PAGE gel (reducing conditions). The matched rabbit polyclonal antibody RP-GranzymeB (/anti-granzyme-b-rabbit-polyclonal-antibody) is raised from the same lab, guaranteeing epitope compatibility. Use RP-GranzymeB at 1:500–1:2000 dilution; refer to its datasheet for the exact validated range. You will see a band at ~32–35 kDa, confirming both antibody performance and expected MW simultaneously — useful when running the antibody against novel tissue or cell-line lysates where endogenous expression is uncertain.

How much recombinant Granzyme B should I load to validate RP-GranzymeB antibody signal linearity on Western blot?

For a linearity/titration blot, load a dilution series of 5, 10, 25, and 50 ng per lane alongside your experimental lysate. This range consistently produces detectable, non-saturating bands with RP-GranzymeB at standard HRP-conjugated secondary antibody dilutions (1:5000 to 1:10,000) and ECL detection. Quantifying band intensity across this series allows you to confirm that the antibody response is linear and to estimate endogenous Granzyme B content in lysates by interpolation. Using the same lot of recombinant as your standards across experiments also reduces inter-assay variability in quantitative Western blot workflows.

How should I store and handle recombinant Granzyme B after receipt to preserve activity?

Upon receipt, store at -20 °C in the single-use aliquots provided. The storage buffer — 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol — stabilizes the enzyme during freeze-thaw, but repeated freeze-thaw cycles progressively reduce specific activity; do not refreeze a thawed aliquot. Thaw on ice and use within the same working day. Avoid diluting to below ~10 µg/mL in buffer lacking a carrier protein; add 0.1% BSA (protease-free) if working at very low concentrations for extended periods. Endotoxin is <0.1 EU/µg by LAL, so the preparation is suitable for cell-based and functional assays without additional depyrogenation.