SPPL-2-C (Recombinant)

Frequently Asked Questions

What molecular weight band should I expect for recombinant SPPL-2-C on SDS-PAGE or Western blot?

SPPL2C has a predicted molecular weight of approximately 75 kDa based on its 684-amino-acid sequence (UniProt Q8IUH8). However, because this recombinant is produced in HEK293 cells, N-linked glycosylation can shift the apparent MW on SDS-PAGE to 80–90 kDa depending on gel percentage and running conditions. We recommend a 10% polyacrylamide gel for clean resolution in this range. If you are running a positive control lane alongside RP-SPPL2C antibody validation, expect the broad band to land above the 75 kDa marker.

Is SPPL-2-C (Recombinant) the full-length protein or a processed catalytic fragment?

This recombinant represents the full-length 684-amino-acid SPPL2C sequence, including both the conserved catalytic YD and GxGD transmembrane motifs characteristic of GxGD-type intramembrane aspartic proteases. It is produced as an active enzyme in HEK293 cells and has not been truncated to an isolated luminal or cytoplasmic domain. The full-length form is relevant for topology-dependent activity assays and for researchers studying the complete multi-pass transmembrane architecture of SPPL2C in spermatogenesis contexts.

What substrates does SPPL-2-C cleave and how do I set up an intramembrane protease activity assay?

SPPL2C is an intramembrane-cleaving aspartic protease (I-CLiP) with substrate specificity overlapping but distinct from SPP, SPPL2A, and SPPL2B. Fluorogenic peptide substrates based on the SPP/SPPL consensus cleavage site (e.g., Boc-AIYLKATS-MCA or custom transmembrane-spanning peptides in detergent micelles) have been used for paralogous enzymes and represent a practical starting point. Activity assays should be performed in detergent-solubilized conditions using 0.1–0.5% DDM or CHAPSO at 37°C in 50 mM sodium acetate pH 6.5–7.0. Include pepstatin A as an aspartyl protease inhibitor control to confirm on-target activity.

What buffer and storage conditions are recommended for SPPL-2-C (Recombinant) activity assays?

The protein is supplied in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol. For activity assays requiring a detergent-solubilized membrane environment, dilute the stock into assay buffer containing 0.1–0.5% CHAPSO or DDM before use — the glycerol carrier is compatible with these detergent systems at concentrations up to 2% in the final reaction. Store unused aliquots at -20°C; do not refreeze after thawing. Repeated freeze-thaw cycles measurably reduce intramembrane protease activity. Purity is >90% by SDS-PAGE and endotoxin is <0.1 EU/µg by LAL, so background from contaminant proteases is minimal.

What starting concentration of SPPL-2-C (Recombinant) should I use for an initial IC50 inhibitor screen?

For intramembrane aspartic protease inhibitor screens with fluorogenic substrates, a common starting point is 50–200 nM recombinant enzyme in the assay well. At these concentrations, signal-to-background ratios are typically sufficient without requiring excessive substrate. Keep final glycerol below 1% and detergent (CHAPSO or DDM) at 0.1–0.25% to avoid suppressing inhibitor binding. Include pepstatin A at 1 µM and an uninhibited vehicle control in each plate. Titrate enzyme concentration in a preliminary linearity experiment to confirm that activity is proportional to protein input before committing to a compound library screen.

Can I use recombinant SPPL-2-C as a positive control for Western blot with the RP-SPPL2C antibody?

Yes — REC-SPPL2C and the matched rabbit polyclonal RP-SPPL2C are validated together for exactly this use case. Load 50–200 ng of recombinant protein per lane alongside your cell lysate samples. Expect a band at approximately 80–90 kDa (glycosylated form) with RP-SPPL2C at a 1:500–1:2000 dilution in standard blocking buffer. Because the antibody and recombinant originate from the same development pipeline, this pairing provides a reliable positive control for antibody lot-to-lot validation, knockdown confirmation, and initial optimization of transfer conditions for this high-MW transmembrane target.

How much SPPL-2-C (Recombinant) should I load per lane when validating RP-SPPL2C antibody dilution series?

For a dilution-series validation of RP-SPPL2C, load a two-fold serial dilution of REC-SPPL2C across lanes: 200, 100, 50, 25, and 12.5 ng per lane. This range lets you determine the lower limit of detection for each antibody dilution tested (typically 1:500 to 1:5000). Run alongside 20–40 µg of testis or HEK293-overexpression lysate as an endogenous/transfected reference. Resolve on a 10% SDS-PAGE gel under reducing conditions. The recombinant's >90% purity means the dominant band at ~80–90 kDa cleanly represents SPPL2C without confounding cross-reactive bands from contaminants.

How should I handle and dilute SPPL-2-C (Recombinant) to avoid loss of activity after thawing?

Aliquots are supplied ready-to-use in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol. Thaw on ice and use within the same working session. Do not refreeze. If you need to dilute for a lower working concentration, use the supplied storage buffer or an equivalent Tris/NaCl buffer supplemented with 0.05% BSA to minimize adsorptive losses at sub-nanomolar concentrations. For detergent-solubilized assays, add the appropriate detergent (DDM or CHAPSO) to the dilution buffer before adding protein. Shelf life is 12 months at -20°C in undisturbed single-use aliquots.