Heat-mediated, ultra-rapid electrophoretic transfer of high and low molecular weight proteins to nitrocellulose membranes.
Here, we report an ultra-rapid method for the transfer of high and low molecular weight proteins to nitrocellulose membranes following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In this procedure, the electro-transfer was performed with heated (70-75 degrees C) normal transfer buffer from which methanol had been omitted. Complete transfer of high and low molecular weight proteins (a purified protein, molecular weight protein standards and proteins from a human tissue extract) could be carried out in 10 min for a 0.75-mm, 7% SDS-PAGE gel. For 10% and 12.5% gels (0.75 mm), the corresponding time was 15 min. In the case of 1.5-mm gels, a complete transfer could be carried out in 20 min for 7%, 10% and 12.5% gels. The permeability of the gel is increased by heat, such that the proteins trapped in the polyacrylamide gel matrix can be easily transferred to the membrane. When the heat-mediated transfer method was compared with a conventional transfer protocol, under similar conditions, we found that the latter method transferred minimal low molecular weight proteins while retaining most of the high molecular weight proteins in the gel. In summary, this procedure is very rapid, avoids the use of methanol and is particularly useful for the transfer of high molecular weight proteins.
Kurien
Biji T
BT
Arthritis and Immunology Program, Oklahoma Medical Research Foundation, 825 NE 13th Street, Oklahoma City, OK 73104, USA. biji-kurien@omrf.ouhsc.edu
Scofield
R Hal
RH
D015153:Blotting, Western / Q000379:methods*; D003101:Collodion / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D006367:HeLa Cells; D006358:Hot Temperature; D006801:Humans; D007700:Kinetics; D008970:Molecular Weight; D011506:Proteins / Q000032:analysis* / Q000737:chemistry
D011506:Proteins; D003101:Collodion