Antibodies, A Laboratory Manual, Second Edition, Edited by Edward A. Greenfield

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Resolving Proteins for Immunoblotting by Gel Electrophoresis

(Protocol summary only for purposes of this preview site)

This protocol describes Tris/glycine SDSpolyacrylamide gel electrophoresis, also known as SDS discontinuous gel electrophoresis or the Laemmli electrophoresis system (Laemmli and Quittner 1974). Here, the stacking and separating monomer solutions are prepared without the reaction initiators (ammonium persulfate [APS] and tetramethylethylenediamine [TEMED]). The gel-casting unit is assembled and tested to make sure that there are no leaks. APS and TEMED are added to the separating monomer solution, and the bottom (separating) gel is poured and allowed to polymerize. The top (stacking) gel is poured, and the comb is inserted to make the wells. The stacking gel is allowed to polymerize and the comb is then removed. The gel cassette is connected to the buffer chambers and the samples are loaded into the wells. The top of the gel is connected to the buffer chamber containing the negative electrode (anode), and the bottom of the gel is immersed into the buffer that makes contact with the positive electrode (cathode). When the electric current is applied, the proteins migrate through the gel toward the anode with a rate that is roughly proportional to the length of their polypeptide chains. The electrophoresis is stopped when the loading dye reaches the bottom of the separating gel. The gel cassette is disassembled, and the proteins are ready for transfer from the gel onto the membrane (Protocols 5 and 6).

Antibodies: A Laboratory Manual, Second edition
Antibodies: A Laboratory Manual, Second edition
Antibodies: A Laboratory Manual, Second edition

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