SPPL-2-B (Recombinant)

Frequently Asked Questions

What is the expected molecular weight of recombinant SPPL-2-B on SDS-PAGE and Western blot?

SPPL2B (UniProt Q8TCT7) is a 592-amino-acid multipass transmembrane protein with a predicted unmodified molecular weight of ~67 kDa. When expressed in HEK293 cells, mammalian glycosylation and other post-translational modifications typically shift the apparent MW to ~70–75 kDa on reducing SDS-PAGE. Run alongside a broad-range protein ladder and expect a diffuse or slightly smeared band consistent with a heavily hydrophobic, glycosylated integral membrane protein. Confirmed purity is >90% by SDS-PAGE under our standard production lot QC.

Which isoform or processing form is this SPPL-2-B recombinant — full-length or a catalytic fragment?

REC-SPPL2B represents the full-length 592-amino-acid human SPPL2B protein, including its GxGD catalytic motif-containing transmembrane core and cytoplasmic N-terminal domain. Unlike gamma-secretase, SPPL2B does not require obligate co-complex partners for initial substrate engagement, so the full-length form is the relevant species for most in vitro biochemical work. The HEK293 expression system preserves the native multipass topology, making this the appropriate form for both cleavage assays and structural binding studies.

What substrates does SPPL-2-B cleave and how do I set up an in vitro cleavage assay?

SPPL2B's two best-characterised substrates are the TNFα C-terminal fragment (TNFα-CTF, the membrane-anchored stub left after TACE/ADAM17 shedding) and ITM2B (BRI2). For a cell-free fluorescent substrate assay, use a commercially available FRET peptide mimicking the TNFα-CTF transmembrane cleavage site. Prepare REC-SPPL2B at 50–200 nM in assay buffer (50 mM sodium acetate pH 4.5–5.5, 0.1% CHAPSO, 0.025% phosphatidylcholine) — note that SPPL2B, like other SPP-family proteases, is optimally active at mildly acidic pH reflecting its endosomal/lysosomal compartment activity. Include a (Z-LL)2-ketone control to confirm GxGD-dependent cleavage.

What buffer conditions work best for SPPL-2-B activity assays — does the standard storage buffer work?

The storage buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol) is appropriate for maintaining enzyme stability and storage, but is not optimal for SPPL2B proteolytic activity. For activity assays, dilute REC-SPPL2B into a pH 4.5–5.5 assay buffer supplemented with 0.1% CHAPSO and a phospholipid such as phosphatidylcholine (0.025%) to mimic the lipid bilayer environment required for intramembrane cleavage. Titrate pH across this range empirically, as peak activity can vary by substrate. Glycerol carryover below 2% final concentration is generally tolerated without significant impact on substrate cleavage kinetics.

What starting concentration of recombinant SPPL-2-B should I use in an inhibitor IC50 assay?

A working range of 50–100 nM REC-SPPL2B is a practical starting point for IC50 determination using FRET-based peptide substrates. At this concentration, substrate turnover is detectable within 30–60 minutes under optimised assay buffer conditions. Keep enzyme concentration at least 10-fold below the Ki of your inhibitor to satisfy the Morrison tight-binding assumption only if needed; otherwise standard Cheng-Prusoff correction from IC50 to Ki applies. Include a (Z-LL)2-ketone reference inhibitor in every plate as an assay validity anchor, with expected IC50 in the low-µM range for SPPL-family enzymes.

Can I use REC-SPPL2B as a positive control for Western blot validation of the matched anti-SPPL-2-B antibody?

Yes — this is a primary intended use of REC-SPPL2B paired with RP-SPPL2B (see /anti-sppl-2b-rabbit-polyclonal-antibody). Load 50–100 ng of REC-SPPL2B per lane on a 10% SDS-PAGE gel. Expect a band at ~70–75 kDa. RP-SPPL2B is a rabbit polyclonal raised against human SPPL2B and is validated for Western blot; using REC-SPPL2B alongside your cell lysate samples provides an unambiguous positive control band at the correct MW, confirming antibody specificity before interpreting endogenous expression data.

How much REC-SPPL2B should I load to get a clean Western blot positive control band without overloading?

Load 50 ng for a strong, clean signal with RP-SPPL2B at a 1:1000 dilution in 5% BSA/TBST. If background is a concern or you are co-running high-protein lysate lanes, titrate between 20–100 ng. Because REC-SPPL2B is >90% pure by SDS-PAGE, even 20 ng should yield a readily detectable band without nonspecific bands confounding interpretation. For chemiluminescent detection, standard 60-second to 5-minute exposures are typically sufficient. Run an unreduced and a reduced lane side-by-side if you are also characterising potential dimeric forms or disulfide-linked artefacts from the transmembrane region.

How should I handle, dilute, and store REC-SPPL2B to maintain activity over time?

REC-SPPL2B is supplied in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol in single-use aliquots, and should be stored at -20°C. Avoid repeated freeze-thaw cycles — activity loss is measurable after as few as two cycles for membrane-topology-dependent intramembrane proteases. Thaw on ice immediately before use. For working dilutions, use your assay buffer and prepare fresh each experiment; do not store diluted enzyme. If extending an experiment across days, prepare aliquots at your working concentration and freeze each separately. Endotoxin is <0.1 EU/µg by LAL assay, suitable for cell-based downstream applications following protease treatment.