Simultaneous electroelution of proteins from denaturing or native gels into a well matrix.
An electroelution device that is based on a semidry blotter and that allows the simultaneous elution of proteins or other charged macromolecules from one-dimensional gels is described. It consists of a central plate that has a matrix of oblong wells arranged in eight columns of 32 wells each, such that when a gel is placed on the plate each lane of bands is underlaid by a column of wells. The plate is placed between the electrodes of a semidry blotter and the wells are sealed by a dialysis membrane resting on polyacrylamide gel block, prior to being filled with transfer buffer. Using radiolabeled molecular weight standards resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, elution is shown to require 30-60 min for 90% or better of proteins between 10 and 120 kDa to be removed from the gel. The recovery volume is 200 microliters per well and losses due to adsorption onto the dialysis membrane are minimal. beta-Galactosidase eluted from a nonreducing, nondenaturing gel was shown to be quantitatively active. The advantages of the device include its ease of assembly and operation, its speed, its reproducibility, and the fact that no gel slicing is required since all proteins are eluted simultaneously, allowing large-scale screening of multiple protein fractions.
Persidis
A
A
University of Cambridge Clinical School, Department of Medicine, Addenbrooke's Hospital, England.
Harcombe
A A
AA
D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D007700:Kinetics; D011489:Protein Denaturation; D001616:beta-Galactosidase / Q000302:isolation & purification* / Q000378:metabolism
D001616:beta-Galactosidase