|ELIMINATION OF METHANOL IN WESTERN BLOTTING|
Western blotting is a technique used by biochemists to electrophoretically transfer proteins from polyacrylamide gels onto a more stable membrane substrate, such as nitrocellulose. The standard conducting solution used during western blotting contains 20% methanol, resulting in the generation of a listed hazardous waste. For many protein transfer applications, particularly those involving high molecular weight proteins, it is possible, and even helpful, to eliminate methanol from the conducting solution.
40 CFR Parts 260-268.
State of Michigan Act 451 Part 111.
Overview of Procedure
The procedure involves placing a protein-containing polyacrylamide gel in direct contact with a piece of nitrocellulose and placing this "sandwich" between two electrodes submerged in a conducting solution. In response to an imposed electric field, the proteins move out of the polyacrylamide gel and onto the surface of the nitrocellulose membrane where the proteins become tightly attached. The resulting nitrocellulose membrane (now a "western blot") contains a replica of the protein pattern found on the polyacrylamide gel. An additional advantage is that proteins adsorbed onto nitrocellulose are more accessible to biochemical reagents than are proteins within polyacrylamide gels.
The standard conducting solution used during western blotting contains 0.192 M glycine, 25 mM trizma base (pH 8.3), and 20% methanol. Due to the solution containing methanol and used in relatively large volumes (up to 3 liters/western blot), the solution represents a significant source of listed hazardous waste.
The problem can be significantly reduced by substituting ethanol or isopropanol for the methanol in the conducting solution. For many protein transfer applications, particularly applications involving high molecular weight proteins, it is possible to eliminate methanol from the conducting solution altogether. When working with larger, high molecular weight proteins, elimination of the methanol results in a significant increase in protein transfer efficiency.
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