Phosphoamino acid analysis of protein immobilized on polyvinylidene difluoride membrane

The direct analysis of phosphorylated proteins bound to polyvinylidene difluoride membrane (PVDFm) has been examined. Use of 14C-methylated marker proteins demonstrated that proteins electroblotted on PVDFm were quantitatively retained through a series of test conditions, which included 1 M hydroxylamine (25 degrees C, 30 min), 0.1 M NaOH (37 degrees C, 30 min), 0.1 M HCl (55 degrees C, 2 h), and 6 U/ml alkaline phosphatase (pH 9.5, 37 degrees C, 24 h). Approximately half the protein remained bound following 2-h treatment in 1 M KOH (55 degrees C). The same series of test conditions were employed to assess the stability of phosphorylated residues in 32P-labeled protein immobilized on PVDFm, in order to assign them as carboxyl-,N-, or O-linked groups. The properties of phosphorylated proteins as determined by this method were comparable to the properties that have been reported for soluble proteins. Use of the PVDFm immobilization step affords simplification of the experimental procedures and permits rapid, quantitative sample recovery using submicrogram quantities of protein. Further, the PVDFm-bound phosphoproteins could be subjected to partial acid hydrolysis directly on the membrane and required no further purification for subsequent identification of the labeled phosphohydroxyamino acids. Definitive identification of labeled phosphoserine residues in histone, phosphoserine and phosphothreonine residues in myelin basic protein and insulin receptor, and phosphotyrosine residues in autophosphorylated insulin receptor was accomplished with as little as 0.2 nCi in about 50 ng of phosphorylated protein.