SPPL-2-A (Recombinant)

Frequently Asked Questions

What is the expected molecular weight of recombinant SPPL-2-A on SDS-PAGE?

Full-length human SPPL2A (UniProt Q8TCT8) has a predicted molecular weight of ~58 kDa, but the HEK293-expressed recombinant typically resolves at 60–65 kDa on reducing SDS-PAGE due to N-linked glycosylation. If you treat the sample with PNGase F prior to electrophoresis, the band shifts down to approximately 58 kDa, confirming glycan contribution. Run alongside a broad-range protein ladder (10–250 kDa). Our lot-release SDS-PAGE confirms >90% purity at this position. A faint higher-molecular-weight smear can appear under non-reducing conditions due to intermolecular disulfide artifacts — use β-mercaptoethanol or DTT in your loading buffer.

Which isoform or processing form of SPPL2A does this recombinant represent?

This recombinant corresponds to the canonical 520-amino-acid isoform 1 (UniProt Q8TCT8-1), which is the predominant endogenous form detected in late endosomal fractions. It is expressed as a full-length construct in HEK293 cells and retains the two-transmembrane-domain topology typical of GxGD aspartyl intramembrane proteases, including the conserved YD and GXGD catalytic motifs. No truncation or soluble ectodomain variant is used. Because it is produced in a mammalian system, the glycosylation pattern and tertiary folding are expected to more closely reflect native enzyme compared to E. coli- or baculovirus-derived preparations.

What substrates does SPPL2A cleave and which are best for in vitro activity assays?

Endogenous SPPL2A substrates include TNFα, FasL (FASLG), ITM2B, CD74, and the foamy virus envelope glycoprotein gp130 — all type II single-pass membrane proteins cleaved within or near the lipid bilayer by regulated intramembrane proteolysis (RIP). For cell-free in vitro assays, membrane-mimetic substrates such as recombinant CD74 transmembrane peptides reconstituted in detergent micelles (CHAPSO or DDM) or bicelles are most commonly used. Fluorogenic peptide substrates spanning the CD74 or TNFα cleavage sites can also be used for continuous kinetic readouts. Confirm cleavage by detecting the released intracellular domain fragment via Western blot.

What buffer conditions work best for SPPL2A protease activity assays in vitro?

Intramembrane aspartyl proteases like SPPL2A require a mildly acidic environment consistent with the late endosomal compartment. Activity assays are typically run in 50 mM sodium acetate or citrate-phosphate buffer, pH 4.5–5.5, supplemented with 0.1–0.25% CHAPSO or 0.05% DDM to solubilize transmembrane substrate peptides. The recombinant is supplied in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol — dilute into assay buffer immediately before use to shift to the active pH range. Include the aspartyl protease inhibitor pepstatin A as a negative control at 1–10 µM to confirm SPPL2A-specific cleavage.

What starting concentration and enzyme-to-substrate ratio should I use for SPPL2A IC50 inhibitor assays?

For IC50 determinations, we recommend starting at 50–200 nM recombinant SPPL2A in the final assay volume, adjusted to remain within the linear range of your substrate turnover curve. Use a substrate concentration at or below the apparent Km (typically 5–20 µM for CD74-derived fluorogenic peptides) to satisfy competitive inhibitor kinetics. Run a substrate saturation curve and a time-course assay at your chosen enzyme concentration first to establish linearity — typically 30–60 min at 37 °C, pH 5.0. Include a DMSO vehicle control (≤0.5% final) and pepstatin A as a reference inhibitor. At least three independent replicates are needed for reliable IC50 fitting.

Can I use recombinant SPPL-2-A as a positive control for Western blot with the matched TPB antibody RP-SPPL2A?

Yes — this recombinant and antibody RP-SPPL2A are produced and validated together in the same lab specifically for this purpose. The antibody is a rabbit polyclonal validated for Western blot detection of SPPL2A. Load 20–50 ng of recombinant SPPL2A per lane on a 10% or 4–20% gradient SDS-PAGE gel under reducing conditions; expect a clean band at 60–65 kDa. This combination is particularly useful when validating antibody RP-SPPL2A against novel cell or tissue lysates, providing an unambiguous size reference. See the antibody product page at /anti-sppl2a-rabbit-polyclonal-antibody for recommended antibody dilutions and blocking conditions.

How much recombinant SPPL-2-A should I load for a Western blot positive control lane?

Load 20–50 ng per lane when using antibody RP-SPPL2A at its recommended working dilution. At 20 ng you should obtain a detectable, clean band at ~60–65 kDa with minimal background; 50 ng gives a robust signal suitable for demonstrating antibody specificity in publications. Avoid loading >100 ng, as this can cause smearing and overloading artifacts that obscure the glycoprotein band. Dilute the recombinant into 1× reducing SDS loading buffer (with DTT or β-mercaptoethanol), boil 5 min at 95 °C, and run alongside your experimental lysates. The protein is supplied at >90% purity by SDS-PAGE, so no additional cleanup is required.

How should I store and handle recombinant SPPL-2-A to maintain activity after receipt?

Upon receipt, briefly spin the vial, then store at −20 °C in the supplied single-use aliquots (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol). The 10% glycerol is critical for cryo-protection — do not dilute it out before freezing. Avoid repeated freeze-thaw cycles; each cycle can reduce activity by 10–20% for membrane-associated proteases. For short-term use (up to 1 week), the protein is stable at 4 °C. When setting up assays, thaw on ice, dilute into assay buffer immediately prior to use, and keep on ice until pipetting. Do not vortex — gentle flicking and brief centrifugation are sufficient to homogenize.