Centrifuge-blotting of proteins after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
A protein transfer method which allows elution and immobilization of polypeptides onto a polyvinylidene difluoride (PVDF) membrane has been developed. The protein band in a gel is eluted by centrifugation. The centrifuge-blotting procedure involves the following steps: (i) visualization of the protein in a sodium dodecyl sulfate (SDS)-polyacrylamide gel with 1 M KCl, (ii) excision of the protein band and equilibration for 15 min in a solution of 0.05% SDS/5% methanol/0.02% dithiothreitol in distilled water, (iii) placing the gel piece in direct contact with the PVDF membrane in the receptacle, (iv) centrifugation at 3000 g for 1 h. A 10 kDa cut-off dialysis membrane is placed beneath the PVDF membrane to retain nonimmobilized protein. The N-terminal sequence of the immobilized protein on the PVDF membrane was determined. For proteins with a molecular mass less than 30 kDa, an overall yield between 10%-30% has been obtained.
Hermansen
L F
LF
Department of Biochemistry, University of Oslo, Norway.
Pedersen
O
O
Sletten
K
K
D002498:Centrifugation; D002919:Chymotrypsinogen / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D008567:Membranes, Artificial; D000432:Methanol; D008970:Molecular Weight; D009211:Myoglobin / Q000302:isolation & purification; D011145:Polyvinyls; D011189:Potassium Chloride; D011506:Proteins / Q000302:isolation & purification*; D017421:Sequence Analysis; D012710:Serum Albumin, Bovine / Q000302:isolation & purification; D005465:Fluorocarbon Polymers
D002919:Chymotrypsinogen; D008567:Membranes, Artificial; D000432:Methanol; D009211:Myoglobin; D011145:Polyvinyls; D011189:Potassium Chloride; D011506:Proteins; D012710:Serum Albumin, Bovine; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers