Anti-ANPR-1 Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight band should I expect for ANPR-1 on Western blot?

ANPR-1 (NPR1/NPR-A) migrates at approximately 130 kDa on reducing SDS-PAGE, corresponding to the full-length single-pass transmembrane receptor. The protein contains an extracellular ligand-binding domain, a single transmembrane helix, and an intracellular guanylate cyclase catalytic domain. Post-translational glycosylation can shift the apparent molecular weight slightly higher than the predicted mass of 120 kDa. In some tissues, particularly kidney lysates, you may observe additional higher-molecular-weight species representing glycosylated or dimeric forms. We recommend including a positive control lysate from human kidney or adrenal tissue where ANPR-1 is abundantly expressed.

What starting dilution do you recommend for Western blot with this ANPR-1 antibody?

Start at 1:1000 dilution for Western blot, as validated in our hands with human tissue lysates. This rabbit polyclonal typically performs well in this range with standard chemiluminescent detection. If signal is weak in your sample type, you can titre up to 1:500. Conversely, if background is high, dilute to 1:2000. Optimal concentration depends on ANPR-1 expression level in your lysate; receptor abundance is highest in kidney medulla, adrenal cortex, and vascular endothelium. Use 5 percent non-fat milk or BSA in TBST for blocking and antibody dilution.

Is this antibody validated for detecting mouse or rat ANPR-1?

This antibody is validated only for human ANPR-1. Cross-reactivity with mouse (Npr1, UniProt P18910) or rat (Npr1, UniProt P18292) is predicted based on sequence homology—human and rodent ANPR-1 share approximately 85 percent identity across the extracellular and intracellular domains. We have not empirically tested reactivity in mouse or rat lysates, so if you are working with rodent samples, we recommend running a pilot experiment with appropriate positive controls such as mouse kidney lysate. Immunogen sequence alignment suggests conserved epitopes, but validation in your system is necessary.

Can I use this ANPR-1 antibody for immunohistochemistry or immunofluorescence?

Yes, this antibody is suitable for IHC and immunofluorescence in addition to Western blot. For IHC on formalin-fixed paraffin-embedded tissue, antigen retrieval is typically required; citrate buffer (pH 6.0) heat-induced epitope retrieval works well for many transmembrane receptors. Start with a 1:100 to 1:200 dilution and optimize based on signal and background in your tissue type. For IF, try 1:50 to 1:100 on fixed cultured cells or frozen sections. Kidney glomeruli and vascular endothelium are good positive-control tissues where ANPR-1 expression is robust and well-characterized.

What sample preparation works best for detecting ANPR-1 in tissue lysates?

ANPR-1 is a transmembrane protein, so use a lysis buffer containing detergent to solubilize membrane fractions effectively. RIPA buffer (with 1 percent NP-40 or Triton X-100, 0.5 percent sodium deoxycholate, 0.1 percent SDS) works well. Include protease and phosphatase inhibitors to preserve receptor integrity and phosphorylation states if you are studying signaling. For tissues rich in ANPR-1—kidney medulla, adrenal gland, or heart atrium—homogenize thoroughly and clarify lysates by centrifugation at 12,000 × g for 10 minutes. Load 20 to 40 micrograms total protein per lane for Western blot.

Does ANPR-1 have splice isoforms I need to account for?

The primary ANPR-1 transcript (NPR1 gene) encodes a 1061-amino-acid protein with no widely annotated functional splice variants in human. Minor splice events have been reported in the literature but are not well-characterized at the protein level. On Western blot, you should see a single predominant band near 130 kDa. If you observe multiple bands, consider the possibility of proteolytic cleavage, differential glycosylation, or cross-reactivity. ANPR-1 can undergo regulated proteolysis in some contexts, releasing extracellular or intracellular fragments, so sample handling and protease inhibition are important.

What are good positive and negative control tissues for ANPR-1 expression?

Positive controls: human kidney (especially medulla), adrenal gland, and vascular endothelium express high levels of ANPR-1. Heart atrial tissue is also positive. For cell lines, human umbilical vein endothelial cells (HUVEC) and some renal epithelial lines express detectable receptor. Negative controls are more challenging because ANPR-1 has broad low-level expression; however, many hematopoietic and lymphoid tissues show minimal expression. A validated negative control is lysate from cells transfected with empty vector alongside ANPR-1-overexpressing cells. Alternatively, compare signal in the presence of blocking peptide if available.

How should I store this antibody and what is the shelf life?

Store the antibody at –20°C as supplied. For routine use, you can aliquot the stock into single-use volumes to avoid repeated freeze-thaw cycles, which may reduce titre and increase aggregation over time. Once thawed, the antibody is stable at 4°C for up to one month when stored in the presence of carrier protein and preservative. Do not store diluted antibody in blocking buffer for more than a few days. Under proper storage at –20°C, polyclonal antibodies typically retain activity for at least two years from the date of manufacture.