Presenilin-1 (Recombinant)

Frequently Asked Questions

What molecular weight does recombinant Presenilin-1 run at on SDS-PAGE and Western blot?

Full-length human PSEN1 has a predicted MW of ~52 kDa (467 aa), but endogenous PSEN1 undergoes constitutive endoproteolysis into an ~28 kDa N-terminal fragment (NTF) and ~20 kDa C-terminal fragment (CTF). Because this recombinant is produced in HEK293 cells under native-like conditions, you may observe both the uncleaved precursor near 52 kDa and the NTF/CTF cleavage products depending on reducing conditions. Under standard denaturing SDS-PAGE, expect the predominant band at ~52 kDa for the full-length form. Running a 10–12% polyacrylamide gel provides adequate resolution of all three forms.

Is recombinant Presenilin-1 the full-length protein or the cleaved NTF/CTF fragments?

The preparation contains full-length PSEN1 (residues 1–467, UniProt P49768) expressed in HEK293 cells. However, because HEK293 cells support native endoproteolytic processing, the preparation may include variable proportions of the ~28 kDa NTF and ~20 kDa CTF — the same species found in the endogenous gamma-secretase complex alongside Nicastrin, APH-1, and PEN-2. If your experiment requires strictly full-length uncleaved PSEN1, confirm the fragment profile by SDS-PAGE before use and account for both species when interpreting Western blot results.

What substrates does Presenilin-1 cleave and how do I measure gamma-secretase activity in vitro?

PSEN1 is the catalytic aspartyl protease subunit of the gamma-secretase complex, mediating intramembrane proteolysis of type I transmembrane substrates including APP (generating Aβ peptides) and Notch (releasing the NICD). For in vitro activity, fluorogenic peptide substrates derived from the APP transmembrane domain (e.g., NMA/DNPK-based quenched peptides) are widely used. Activity requires assembly of the full holocomplex; isolated recombinant PSEN1 alone has minimal catalytic activity. Use this recombinant primarily as an antigen standard, immunoprecipitation positive control, or antibody validation reference rather than a standalone enzyme for cleavage assays.

What buffer should I use when setting up assays with recombinant Presenilin-1?

This recombinant is supplied in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol — a standard stabilizing buffer suitable for immunoprecipitation and Western blot calibration applications. For functional reconstitution experiments, note that gamma-secretase activity is typically assessed in detergent-solubilized membrane preparations (e.g., CHAPSO or DDM) supplemented with phosphatidylcholine/phosphatidylethanolamine lipids; the supplied buffer alone is not optimized for intramembrane proteolysis assays. Avoid diluting below the stability threshold without carrier protein (0.1% BSA is a common supplement). Do not add reducing agents at high concentrations, as these can disrupt the aspartyl protease active site.

What starting concentration of recombinant Presenilin-1 should I use for antibody validation or IP positive control?

For Western blot antigen standard use, 50–200 ng per lane provides a strong, reproducible signal with most anti-PSEN1 antibodies, including the matched TPB rabbit polyclonal (RP-Presenilin1). For immunoprecipitation positive control, a starting input of 0.5–1 µg per reaction is sufficient when using standard Protein A/G bead protocols. Titrate down from 200 ng on your first Western blot run to identify the linear detection range for your specific antibody concentration and chemiluminescence system. Because purity is >90% by SDS-PAGE and endotoxin is <0.1 EU/µg, background contributions from contaminants are minimal.

Can I use recombinant Presenilin-1 as a positive control for Western blot with the RP-Presenilin1 antibody?

Yes — this is a validated use case. The matched rabbit polyclonal antibody RP-Presenilin1 (/anti-presenilin-1-rabbit-polyclonal-antibody) was produced and validated in the same laboratory as this recombinant, ensuring antigen-antibody compatibility. Load 50–100 ng of recombinant PSEN1 per lane alongside your cell lysate samples; the antibody should detect the ~52 kDa full-length band (and potentially NTF/CTF fragments) under standard PVDF or nitrocellulose transfer conditions. This pairing eliminates ambiguity about whether a negative result in your lysate reflects poor antibody performance versus true target absence.

How much recombinant Presenilin-1 should I load for a Western blot positive control lane?

Load 50–150 ng per lane as a starting point. At 50 ng, the ~52 kDa full-length PSEN1 band is typically detectable with the matched RP-Presenilin1 rabbit polyclonal at a 1:1,000–1:2,000 dilution using standard HRP-conjugated secondary antibodies and ECL detection. If you are using a different anti-PSEN1 antibody, begin at 100 ng to account for variable sensitivity. Running a 3-point dilution series (50, 100, 200 ng) on your first blot establishes a working curve and confirms linearity within your detection system.

How should I store recombinant Presenilin-1 and what is the shelf life after aliquoting?

Store at -20°C in single-use aliquots immediately upon receipt. The supplied buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol) provides adequate cryoprotection at -20°C. Avoid repeated freeze-thaw cycles — each cycle can cause measurable aggregation and activity loss for membrane-associated proteins like PSEN1. Properly stored aliquots are stable for up to 12 months from the date of manufacture. Once thawed, keep on ice and use within the same working session. Do not refreeze a thawed aliquot. For long-term archival storage beyond 12 months, -80°C is recommended.