Acid and base hydrolysis of phosphoproteins bound to immobilon facilitates analysis of phosphoamino acids in gel-fractionated proteins.
Immobilon, a membrane of polyvinylidene difluoride to which gel-fractionated proteins can be transferred electrophoretically, was found to be an excellent matrix for the analysis of the phosphoamino acid content of phosphoproteins. Hydrolysis of 32P-labeled proteins bound to Immobilon with 5.7 N HCl resulted in the release of 90% of the 32P in the form of Pi, phosphoamino acids, and phosphopeptides. Two-dimensional electrophoretic analysis of the released phosphoamino acids yielded undistorted patterns. Because direct hydrolysis of proteins transferred to Immobilon eliminated the need for both preparative extraction of proteins from a gel and recovery by precipitation, analysis was rapid and yields of phosphoamino acids were extremely consistent. The yield of phosphoamino acids from proteins bound to Immobilon, unlike that from proteins eluted from gels, was independent of the size of the protein. The detection of 32P-labeled, phosphotyrosine-containing proteins in sodium dodecyl sulfate-polyacrylamide gels has been shown to be substantially improved by incubation of the gel in 1.0 N KOH for 2 h at 55 degrees C. Base hydrolysis of proteins bound to Immobilon proved to be faster and more sensitive than hydrolysis of proteins in gels. Less than 10% of bound protein was lost from Immobilon during the 2-h incubation at 55 degrees C in 1.0 N KOH. The autoradiographic image after alkaline hydrolysis of proteins on Immobilon was sharper than that obtained after hydrolysis of proteins in the gel. In addition, unlike base-treated gels, the dimensions of the Immobilon filter were unaffected by treatment with base.(ABSTRACT TRUNCATED AT 250 WORDS)
Kamps
M P
MP
Molecular Biology and Virology Laboratory, Salk Institute, San Diego, California 92138.
Sefton
B M
BM
D000596:Amino Acids / Q000032:analysis*; D000818:Animals; D001358:Avian Sarcoma Viruses / Q000235:genetics; D002472:Cell Transformation, Viral; D002478:Cells, Cultured; D002846:Chromatography, Affinity / Q000379:methods; D002850:Chromatography, Gel / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007202:Indicators and Reagents; D051379:Mice; D008807:Mice, Inbred BALB C; D010750:Phosphoproteins / Q000096:biosynthesis / Q000302:isolation & purification*; D010761:Phosphorus Radioisotopes; D011145:Polyvinyls; D011865:Radioisotope Dilution Technique; D005465:Fluorocarbon Polymers
D000596:Amino Acids; D007202:Indicators and Reagents; D010750:Phosphoproteins; D010761:Phosphorus Radioisotopes; D011145:Polyvinyls; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers