ANPR-1 (Recombinant)

Frequently Asked Questions

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

The unmodified polypeptide chain of human ANPR-1 (NPR1, 1,061 aa) predicts a molecular weight of approximately 119 kDa. Because this recombinant is produced in HEK293 cells, N-linked glycosylation adds roughly 15–25 kDa, so the predominant band typically migrates at 135–145 kDa under reducing SDS-PAGE conditions. Run a 6–8% polyacrylamide gel for clean resolution in this range. Under non-reducing conditions, aberrant migration is possible due to intact disulfide bonds in the extracellular domain. Always include a broad-range molecular weight ladder spanning 100–250 kDa.

Which isoform or processing form of ANPR-1 does the recombinant protein represent?

This recombinant corresponds to the canonical full-length human NPR1 isoform (UniProt P16066-1, residues 1–1,061), including the signal peptide, extracellular ligand-binding domain, single transmembrane segment, kinase homology domain, hinge region, and C-terminal guanylate cyclase catalytic domain. The signal peptide (residues 1–22) is processed co-translationally in HEK293 cells, so the mature secreted/membrane form begins at residue 23. No alternative splice isoforms with truncated cyclase domains are present in this preparation.

What substrate and assay format should I use to measure ANPR-1 guanylate cyclase activity in vitro?

ANPR-1 catalyses conversion of GTP to cyclic GMP (cGMP; EC 4.6.1.2). For in vitro activity assays, use 1 mM GTP as substrate in a reaction buffer of 50 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM DTT, 0.5 mM IBMX (to inhibit phosphodiesterases). Quantify cGMP production with a competitive ELISA kit or HTRF cGMP assay after 30–60 min at 37 °C. A starting enzyme concentration of 50–200 nM recombinant ANPR-1 is recommended for initial titration. Verify activity is stimulated ≥3-fold by addition of 100 nM ANP (NPPA) ligand.

How should I optimize buffer conditions and ANP concentration for ANPR-1 activity assays?

The supplied storage buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol) is compatible with most activity assays after a simple dilution step. Dilute into assay buffer to reduce glycerol below 1% final concentration, as higher glycerol can suppress cGMP signal. Mg²⁺ is essential — confirm 5–10 mM MgCl₂ in the final reaction. For dose–response experiments, titrate synthetic ANP across 0.01–1,000 nM; reported EC₅₀ values for NPR1 activation by ANP fall in the 1–10 nM range. Include a vehicle-only (no ligand) baseline and a saturating ligand control in every experiment.

Can I use recombinant ANPR-1 as a positive control for Western blot with TPB antibody RP-ANPR1?

Yes — recombinant ANPR-1 (REC-ANPR1) is the validated positive control for the matched rabbit polyclonal antibody RP-ANPR1 (/anti-anpr-1-rabbit-polyclonal-antibody). Load 20–50 ng of recombinant protein per lane on a 6–8% SDS-PAGE gel under reducing conditions. The glycosylated band at 135–145 kDa should resolve cleanly with RP-ANPR1 at a primary antibody dilution of 1:500–1:2,000. Because REC-ANPR1 and RP-ANPR1 are produced in the same laboratory, lot-to-lot epitope compatibility is guaranteed, eliminating a common source of validation uncertainty.

How much recombinant ANPR-1 should I load for Western blot positive control to avoid signal saturation?

Start with a three-point titration: 10 ng, 25 ng, and 50 ng per lane. With RP-ANPR1 at 1:1,000 and a standard HRP-conjugated anti-rabbit secondary (1:5,000–1:10,000), 25 ng typically gives a clean, unsaturated band at 135–145 kDa on ECL detection with a 30–60 second exposure. If you are running the recombinant alongside cell lysate lanes, load the 10 ng point in the same blot to keep relative signal in a comparable range. Avoid loading >100 ng, which tends to produce a smeared high-MW artifact from aggregation.

How should I handle, dilute, and store recombinant ANPR-1 to maintain activity?

REC-ANPR1 is supplied as a ready-to-use solution in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol at >90% purity by SDS-PAGE and <0.1 EU/µg endotoxin (LAL). Store at −20 °C in single-use aliquots; repeated freeze-thaw cycles measurably reduce guanylate cyclase activity. When diluting for assays, use carrier protein (0.1% BSA) in the dilution buffer to minimize adsorptive losses at low concentrations (<10 nM). Working aliquots can be kept at 4 °C for up to 48 hours. Do not dilute into buffers below pH 6.5 or above pH 8.5.

Is recombinant ANPR-1 suitable for SPR or pull-down binding studies with ANP or BNP peptides?

Yes. Because REC-ANPR1 is produced in HEK293 cells, the extracellular ligand-binding domain carries native N-linked glycosylation and correct disulfide bonding, both of which are required for high-affinity natriuretic peptide binding. For SPR, immobilize REC-ANPR1 via amine coupling at 5–10 µg/mL in 10 mM sodium acetate pH 4.5, and flow ANP or BNP as analyte. Reported K_D values for ANP binding to NPR1 ECD are in the low nanomolar range (1–5 nM). For pull-down experiments, RP-ANPR1 (SKU: RP-ANPR1) can be used to confirm capture of the recombinant bait protein by Western blot.