Presenilin-2 (Recombinant)

Frequently Asked Questions

What molecular weight does recombinant Presenilin-2 run at on SDS-PAGE and Western blot?

Full-length human PSEN2 has a predicted molecular weight of ~50 kDa, but endoproteolytic processing generates an N-terminal fragment (NTF, ~28–30 kDa) and a C-terminal fragment (CTF, ~20–24 kDa). Under denaturing SDS-PAGE conditions, you will typically observe both processed fragments rather than a dominant full-length band, reflecting the near-complete autoproteolysis that occurs co-translationally in HEK293 cells. Confirm band identity with our matched antibody RP-Presenilin2, which is validated to recognize both NTF and CTF on Western blot.

Is this recombinant Presenilin-2 the full-length protein or a processed fragment?

The recombinant is expressed from the full-length 448-amino-acid human PSEN2 open reading frame (UniProt P49810) in HEK293 cells. However, PSEN2 undergoes obligate endoproteolytic autocleavage during biogenesis, so the final product contains the NTF and CTF held in non-covalent association — the same configuration found in the endogenous active γ-secretase holocomplex. This makes it biochemically representative of the catalytically competent form, not an artificially truncated construct.

What substrates does recombinant Presenilin-2 cleave in a cell-free γ-secretase activity assay?

As the catalytic aspartyl protease subunit of γ-secretase, PSEN2 processes type I transmembrane substrates including the C99 and C83 fragments of amyloid precursor protein (APP) and Notch intracellular domain (NICD) precursors. For in vitro activity assays, a commonly used substrate is a fluorogenic APP-derived peptide (e.g., MLSF-based FRET peptides) or recombinant APP-C99 fragment. Proteolytic activity produces Aβ peptides (Aβ40/Aβ42 for APP). Validate cleavage products by Aβ ELISA or mass spectrometry alongside appropriate γ-secretase inhibitor controls such as DAPT or L-685,458.

What buffer conditions are recommended for Presenilin-2 recombinant activity assays?

The protein is supplied in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol — conditions compatible with most cell-free γ-secretase assay formats. For activity assays, dilute into assay buffer containing 50 mM sodium acetate or HEPES pH 7.0–7.4, supplemented with 0.1–0.25% CHAPSO or DDM detergent to maintain membrane protein solubility. Avoid strong ionic detergents (SDS, Triton X-100 above 0.1%) and reducing agents above 1 mM DTT, as these disrupt the NTF–CTF heterodimer association critical for catalytic activity.

What is a recommended starting concentration for Presenilin-2 in an in vitro γ-secretase cleavage assay?

A practical starting point is 50–200 nM recombinant PSEN2 (as part of a reconstituted or semi-purified γ-secretase complex) with substrate concentrations in the 1–10 µM range, assaying for 2–4 hours at 37°C. Because PSEN2 functions within the multi-subunit complex, activity in isolation from nicastrin, APH-1, and PEN-2 may be reduced; consider supplementing with native membrane fractions or co-expressing complex partners. Titrate enzyme from 25–500 nM to establish linearity before running inhibitor IC50 experiments.

How do I use recombinant Presenilin-2 as a positive control for Western blot with the RP-Presenilin2 antibody?

Load 50–200 ng of recombinant PSEN2 per lane on a 12–15% SDS-PAGE gel. After transfer, probe with RP-Presenilin2 (/anti-presenilin-2-rabbit-polyclonal-antibody) at a 1:500–1:2,000 dilution in 5% non-fat milk/TBST. Expect bands at ~28–30 kDa (NTF) and ~20–24 kDa (CTF). This recombinant and antibody originate from the same production pipeline, so band identity is guaranteed to match, making this combination a reliable positive control for antibody validation experiments or when establishing PSEN2 detection in novel tissue lysates.

How much recombinant Presenilin-2 should I load as a Western blot positive control alongside cell lysate?

For a clean, non-overloaded positive control lane, 100 ng is a reliable starting point. At this quantity, both the NTF (~28–30 kDa) and CTF (~20–24 kDa) bands are detectable with RP-Presenilin2 without saturating the signal. If your cell lysate lanes contain very low PSEN2 expression (e.g., non-neuronal lines), you may drop to 50 ng to keep the positive control lane intensity comparable. Run the recombinant in a dedicated lane separate from lysate to avoid cross-contamination and confirm expected mobility against your molecular weight ladder.

How should I store and handle recombinant Presenilin-2 to maintain activity and avoid degradation?

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 cryoprotection, but repeated freeze-thaw cycles progressively disrupt the NTF–CTF heterodimer and reduce activity — avoid more than one freeze-thaw per aliquot. On ice, working aliquots are stable for up to 8 hours. For dilutions below ~10 nM, add 0.1% BSA (protease-free) as a carrier to minimize adsorptive losses on tube walls. Purity is >90% by SDS-PAGE; endotoxin is confirmed <0.1 EU/µg by LAL assay.