General Western Blot Methods Protocols

Immunoblotting: Preparing Cell Lysates Protocol For immunoblotting experiments, it is often important to compare the total amount of an antigen from many different sources or to learn if a particular source has the antigen under study.  In the approach described here, tissue cultures, bacteria, yeast cells, tissues, and other sources of antigens are disrupted directly in an electrophoresis sample .

Immunoblotting: Preparing Protein Solutions Protocol Protocol describes how proteins are mixed with a sample buffer and prepared for electrophoresis on standard Tris/glycine SDS-polyacrylamide gels.

Immunoblotting: Semi-Dry Electrophoretic Transfer of Proteins from Gels to Membranes Protocol The transfer of proteins from a Tris/glycine SDS-polyacrylamide gel to a membrane using a semi-dry method is achieved by placing the gel next to a piece of nitrocellulose filter.  This sandwich is placed directly between two plate electrodes, and the proteins are then transferred from the gel onto the filter.

Immunoblotting: Submerged Electrophoretic Transfer of Proteins from Gels to Membranes Protocol Transfer of proteins from a Tris/glycine SDS-polyacrylamide gel to a membrane using the submerged method is achieved by placing the gel next to a piece of nitrocellulose filter, submerging this sandwich in a large volume of transfer buffer in a transfer tank, and running current from one side of the transfer tank to another.  The proteins are then eluted by transferring them from the gel onto the filter.

Western Blot Protocol This protocol was developed for the BIORAD protein gel and transfer apparatus.  The buffers can be used with any electrophoresis/transfer system.