Generation of polyclonal antibodies against purified rat whey acidic proteins and the synthesis of a tracer fusion protein suitable for use in radioimmunoassays.
Rodent milk consists mainly of caseins and whey proteins. A major component of the latter group is the whey acidic proteins (WAP) the gene for which has been cloned recently and shown to contain several potential glucocorticoid receptor binding sites. Studies on the regulation of this gene by glucocorticoids would be greatly enhanced by the availability of a radioimmunoassay for WAP. Rat milk was obtained from lactating Sprague-Dawley rats and the WAP purified to homogeneity by ammonium sulphate fractionation, gel filtration and ion exchange chromatography. Rabbit polyclonal antibodies were raised against the purified WAP and used to probe a Western blot of whey proteins. The major band recognized by the antibody corresponded in molecular weight to purified WAP. Problems associated with radiolabelling the tyrosine-free WAP molecule necessitated the fusion of a tyrosine containing protein with the rat milk protein. A rat WAP cDNA clone was ligated to the glutathione transferase gene, the fusion protein expressed in Escherichia coli and purified by a one-step procedure on a glutathione affinity column. Purified WAP readily displaced the radiolabelled recombinant tracer in a radioimmunoassay.
Krozowski
Z
Z
Medical Research Centre, Prince Henry's Hospital, Melbourne, Victoria, Australia.
D000818:Animals; D000906:Antibodies / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism; D015153:Blotting, Western; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D002853:Chromatography, Liquid / Q000379:methods; D003001:Cloning, Molecular; D005260:Female; D005982:Glutathione Transferase / Q000276:immunology* / Q000378:metabolism; D008894:Milk Proteins / Q000276:immunology* / Q000378:metabolism; D011863:Radioimmunoassay; D051381:Rats; D011919:Rats, Inbred Strains
D000906:Antibodies; D008894:Milk Proteins; C024471:whey acidic proteins; D005982:Glutathione Transferase