A semidry electroblotting system efficiently transfers both high- and low-molecular-weight proteins separated by SDS-PAGE.
A general procedure is described for the simultaneous recovery by electroblotting on nitrocellulose or polyvinylidene difluoride (PVDF) membranes of both high- and low-molecular-weight proteins already separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It is based on the use of an original set of buffers which creates a stable pH boundary between the two faces of the blotted membrane. The gel to be transferred is placed on the basic side (pH 8.4) of this boundary, which also contains sodium dodecyl sulfate. Under these conditions even the high-molecular-weight proteins are efficiently eluted out of the acrylamide. Their migration is then stopped on the membrane, due to the change of pH with the acid side (pH 3.8) of the boundary, and the presence of methanol in it. This increases the adsorption on the membrane of the low-molecular-weight polypeptides which have a low affinity for nitrocellulose or PVDF. The procedure permits a quantitative transfer of complex samples which contain poorly soluble proteins or with molecular masses outside the range of 20-70 kDa, like grain storage proteins of cereals.
Laurière
M
M
Laboratoire de Chimie Biologique, centre INRA de Grignon, Thriveral-Grignon, France.
D002021:Buffers; D003101:Collodion; D004586:Electrophoresis / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D005069:Evaluation Studies as Topic; D001467:Hordeum / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D008567:Membranes, Artificial; D008970:Molecular Weight; D010940:Plant Proteins / Q000737:chemistry / Q000302:isolation & purification; D011145:Polyvinyls; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000592:standards; D012015:Reference Standards; D005465:Fluorocarbon Polymers
D002021:Buffers; D003101:Collodion; D008567:Membranes, Artificial; D010940:Plant Proteins; D011145:Polyvinyls; D011506:Proteins; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers