Magnesium as a cofactor is required in many cellular activities. Mg+2 reabsorption in the kidney is mediated primarily be a poorly understood paracellular pathway (passage of solutes between the cells) in the thick ascending limb of Henle (TAL). Tight junctions constitute the barrier to paracellular conductance. Familial hypomagnesaemia with hypercalciuria and nephrocalcinosis (FHHNC, MIM 248250) is a complex renal tubular disorder characterized by hypomagnesaemia, hypercalciuria, advanced nephrocalcinosis, hyposthenuria and progressive renal failure. The mode of inheritance is autosomal recessive. A primary defect in the reabsorption of magnesium in the TAL has been proposed to be essential in FHHNC pathophysiology. Recently, mutations in the gene paracellin-1 (PCLN-1/Claudin-16) have been identified as the underlying genetic defect in FHHNC. Null mutation of PCLN-1/Claudin-16 has been shown to produce chronic interstitial nephritis in cattle. PCLN-1 gene codes for a protein of 305 aa (chromosome 3q27), with four TM domains and intracellular NH2- and COOH-termini. PCLN-1 belongs to the Claudin family of proteins. It is 10-18% related with claudins. PCLN-1 is only expressed in tight junction of TAL implicated in Mg+2-reabsorption.

Source of Antigen and Antibodies:

A 17 aa peptide sequence (designated as PCLN11-P, control peptide) within the cytoplasmic, C-terminus of human PCLN-1 (1) was selected for antibody production. This region is least conserved in various actin isoforms. The peptide was coupled to KLH, and antibodies generated in rabbits. Control peptide was also coupled to Sepharose and use for affinity purification of antibodies.

Recommended Usage:

Western Blotting (1:1-:1:3K) using ECL technique.

ELISA: Control peptide can be used to coat ELISA plates at 1 ug/ml and detected with antibodies (1:10-50K for neat serum and 0.5-1 ug/ml for affinity pure).

Histochemistry & Immunofluorescence: Not tested. We recommend the testing of affinity pure antibodies (2-10 ug/ml) for this purpose.

Specificity & Cross-reactivity:

The human PCLN11-P is 70% conserved in bovine PCLN-1. Antibody cross-reactivity in various species has not been studied. Control peptide, because of its small size, is not suitable for Western. It is recommed for ELISA, dot blot, or for antibody blocking experiments (use 5-10 ug of control peptide per 1 ug of aff pure IgG or 1 ul of antiserum) to demonstrate antibody specificity (see below).

References:

(1). Simon DB et al (1999) Science 285, 103-106; Wong V and Goodenough DA (1999) Science 285, 62; Ohba Y et al (2000) Genomics 68, 229-36; Weber S et al (2000) Eur J Hum Genet 8, 414-22;