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 P-32-labeled proteins from dried p olyacrylamide gels to small disks of nitrocellulose, nylon or polyviny lidene difluoride (PVDF) is described. Gel pieces containing the desir ed phosphoprotein are rehydrated in buffer containing sodium dodecyl s ulfate (SDS)and sealed in agarose in a glass tube over a supporting ge l of polyacrylamide. Protein is transferred upwards through a disconti nuous density gradient of SDS-buffer and methanol to a disk of membran e sealed to the mouth of the tube with dialysis membrane. The method a llows the concentration of a phosphoprotein present in several gel pie ces to a single disk of immobilized membrane. Recovery of phosphoprote in was at least as good as obtained with conventional electroblotting. Application of the method to the analysis of the phosphoamino acid co ntent of the astrocyte marker, glial fibrillary acidic protein, is des cribed.