Premium
Electrotransfer of fixed phosphoproteins from pieces of dried polyacrylamide gel to small disks of nitrocellulose, nylon or polyvinylidene difluoride
Author(s) -
Gonçalyes Carlos A.,
Rodnight Richard
Publication year - 1993
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.11501401123
Subject(s) - nitrocellulose , agarose , chromatography , polyacrylamide , electroblotting , membrane , phosphoprotein , chemistry , sodium dodecyl sulfate , polyacrylamide gel electrophoresis , trypsin , buffer (optical fiber) , biochemistry , polymer chemistry , telecommunications , phosphorylation , computer science , enzyme
A simple method for the transfer of 32 P‐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.