AEBP-1 (Recombinant)

Frequently Asked Questions

What is the expected molecular weight of recombinant AEBP-1 on SDS-PAGE and Western blot?

AEBP-1 is a 1,158-amino-acid protein with a predicted unmodified MW of ~130 kDa. Because REC-AEBP1 is expressed in HEK293 cells — a system that supports mammalian glycosylation — the observed band on reducing SDS-PAGE typically migrates at 140–160 kDa due to N-linked glycosylation. On Western blot under denaturing conditions, expect a diffuse band in the 140–160 kDa range; this smearing is characteristic of heavily glycosylated secreted proteins and is not indicative of degradation. Purity is confirmed at >90% by SDS-PAGE prior to release.

Is recombinant AEBP-1 the full-length protein or a processed fragment? Does it include the signal peptide?

REC-AEBP1 is produced as full-length AEBP-1 (residues 1–1158, UniProt Q8IUX7) expressed in HEK293 cells. The endogenous N-terminal signal peptide directs the nascent protein through the secretory pathway, where it is co-translationally cleaved; the recombinant product collected from conditioned medium therefore represents the mature, processed form without the signal peptide. No additional propeptide or tag truncation is introduced, so the carboxypeptidase-like domain is intact and present in the final product, which is relevant for fibrillogenesis and ECM-binding assays.

What substrate or assay can I use to confirm AEBP-1 carboxypeptidase-like activity in vitro?

AEBP-1 carries a carboxypeptidase-like domain but lacks the catalytic zinc-coordinating residues found in classical metallocarboxypeptidases, meaning it does not exhibit conventional carboxypeptidase hydrolytic activity against standard substrates such as hippuryl-Phe or furylacryloyl-Ala-Phe. Functional characterization is better pursued through collagen fibrillogenesis assays: measure fibril assembly kinetics (turbidity at A313 nm) in the presence of type I collagen (0.1–0.5 mg/mL) and titrated REC-AEBP1 (0.1–2 µg/mL) in 50 mM Tris-HCl pH 7.5, 150 mM NaCl at 37°C. AEBP-1 accelerates fibril formation, providing a quantifiable functional readout.

What buffer conditions are recommended for AEBP-1 fibrillogenesis or binding assays?

REC-AEBP1 is supplied in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol. For collagen fibrillogenesis assays, dilute the recombinant into assay buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl) to keep glycerol below 1% final concentration, as higher glycerol can independently influence fibril kinetics. For pull-down or SPR binding experiments with ECM components, PBST (pH 7.4, 0.05% Tween-20) is compatible. Avoid detergents above 0.1% and pH below 6.5, which can reduce protein solubility. A starting concentration of 0.5–1 µg/mL is a practical titration point for initial dose-response experiments.

What starting concentration of recombinant AEBP-1 should I use for a cell-based ECM or fibroblast stimulation assay?

For cell-based assays — such as stimulating fibroblasts or mesenchymal cells to probe AEBP-1's role in ECM remodeling — a starting concentration of 100–500 ng/mL in serum-free or low-serum media is a reasonable range, consistent with concentrations used for other secreted ECM regulators. Endotoxin levels for REC-AEBP1 are <0.1 EU/µg by LAL assay, so endotoxin-driven inflammatory artifacts are minimized. Titrate in 3-fold dilutions from 500 ng/mL downward and confirm cellular response (e.g., collagen gel contraction, fibril deposition by immunofluorescence) at 24–72 hours post-treatment.

Can I use REC-AEBP1 as a positive control for Western blot with the matched anti-AEBP-1 antibody RP-AEBP1?

Yes — this is one of the primary intended uses of REC-AEBP1. The matched rabbit polyclonal antibody RP-AEBP1 (/anti-aebp-1-rabbit-polyclonal-antibody) is validated for Western blot against this exact recombinant in-house, so band identity at 140–160 kDa is confirmed. Load 50–200 ng of REC-AEBP1 per lane alongside your cell lysate or conditioned medium samples. This quantity gives a clean, strong signal without overloading, and serves as both a molecular weight reference and an antibody sensitivity benchmark within the same blot. Dilute REC-AEBP1 in 1× Laemmli sample buffer and boil 5 min before loading.

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

Load 50–200 ng of REC-AEBP1 per lane for a Western blot positive control. At 50 ng, the band at 140–160 kDa is detectable with RP-AEBP1 at a 1:1,000 dilution using standard HRP-conjugated secondary antibodies and chemiluminescent detection. If you are calibrating a new lot of RP-AEBP1 or comparing detection sensitivity across antibody dilutions, running a 3-point titration (50, 100, 200 ng) in adjacent lanes is practical. Avoid loading more than 500 ng, as over-signal and blooming can obscure migration behavior of the glycosylated band.

How should I store and handle REC-AEBP1 to preserve activity, and what is its shelf life after thawing?

Store REC-AEBP1 at -20°C in the single-use aliquots provided. Avoid repeated freeze-thaw cycles — each cycle can reduce soluble protein yield and functional activity, particularly relevant for a secreted ECM regulator like AEBP-1 that relies on tertiary structure for collagen interactions. Once thawed, keep on ice and use within 4–8 hours; do not refreeze. The protein is stable for at least 12 months from date of receipt at -20°C when stored undisturbed. Working dilutions in assay buffer (without glycerol) should not be stored; prepare fresh from the frozen stock for each experiment.