Electrotransfer of fixed phosphoproteins from pieces of dried polyacrylamide gel to small disks of nitrocellulose, nylon or polyvinylidene difluoride.
A simple method for the transfer of 32P-labeled proteins from dried polyacrylamide gels to small disks of nitrocellulose, nylon or polyvinylidene difluoride (PVDF) is described. Gel pieces containing the desired phosphoprotein are rehydrated in buffer containing sodium dodecyl sulfate (SDS) and sealed in agarose in a glass tube over a supporting gel of polyacrylamide. Protein is transferred upwards through a discontinuous density gradient of SDS-buffer and methanol to a disk of membrane sealed to the mouth of the tube with dialysis membrane. The method allows the concentration of a phosphoprotein present in several gel pieces to a single disk of immobilized membrane. Recovery of phosphoprotein was at least as good as obtained with conventional electroblotting. Application of the method to the analysis of the phosphoamino acid content of the astrocyte marker, glial fibrillary acidic protein, is described.
Gonçalves
C A
CA
Departamento de Bioquímica, UFRGS, Porto Alegre, RS Brazil.
Rodnight
R
R
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D003101:Collodion; D004591:Electrophoresis, Polyacrylamide Gel; D005904:Glial Fibrillary Acidic Protein / Q000302:isolation & purification; D006624:Hippocampus / Q000737:chemistry; D047908:Intracellular Signaling Peptides and Proteins; D007700:Kinetics; D008565:Membrane Proteins; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D009757:Nylons; D010750:Phosphoproteins / Q000302:isolation & purification*; D011145:Polyvinyls; D011506:Proteins / Q000302:isolation & purification; D051381:Rats; D017208:Rats, Wistar; D016704:Synapsins / Q000302:isolation & purification; D005465:Fluorocarbon Polymers
D000596:Amino Acids; D003101:Collodion; D005904:Glial Fibrillary Acidic Protein; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D009757:Nylons; D010750:Phosphoproteins; D011145:Polyvinyls; D011506:Proteins; D016704:Synapsins; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers