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PAGE is a laboratory technique designed to separate proteins according to their size, shape, and also charge. Proteins for PAGE are usually prepared by placing them in an anionic (negative charge) detergent mixture, which, along with heating to approximately 60°C, denatures the protein, breaks any cysteine–cysteine disulfide bonds, and creates a fairly linear protein structure. PAGE can also be run with an addition of SDS. SDS, if used, is included in the detergent mixture and assists in disrupting the proteins’ secondary and tertiary structures. In addition, SDS binds to each peptide chain in a ratio of one SDS per two amino acid residues, thereby adding a negative charge proportional to the peptide length. The addition of SDS to PAGE samples, therefore, also makes the proteins’ shapes and native charges irrelevant; only the total length and the resulting SDS charge matters.

Polyacrylamide gel is composed of linear acrylamide molecules, cross-linked by bisacrylamide via the catalytic actions of ammonium persulfate and tetramethylethylenediamine (TMED). Cross-linking forms a web-like polyacrylamide lattice with pores of approximately the same size throughout. Varying the amounts of acrylamide, bisacrylamide, and aqueous solution allows scientists to accurately vary the pore size and, therefore, the relative ability to separate differently sized protein molecules.

After polymerization, the gel is placed in an electrophoresis device and immersed in a buffer with a cathode (positive charge) at the top and an anode (negative charge) at the bottom. The protein samples are “loaded” onto individual gel lanes formed by a removable comb. A tracking dye is usually included in the samples to monitor the progress of the proteins through the gel.

The electric field causes the negatively charged proteins (charge usually enhanced by the proportional negative charge of SDS molecules) to move toward the anode proportional to their length (and, therefore, charge). Movement through the gel is not directly proportional to the overall charge, though, because the larger proteins will encounter much more difficulty in moving through the pores because of their size and linear rigidity. Although the length of each peptide increases the electrical force moving it down the gel, the “filtering” action of the gel pores will allow smaller molecules to travel further down the gel, whereas larger molecules remain near the top.

Basis of PAGE Separation

(A) The PAGE system employs the electric charge deployed between a positive electrode (anode) and negative electrode (cathode). (B) Proteins are loaded (origin) onto polyacrylamide gel, which is exposed to this electric field. Negatively charged proteins are driven toward the anode, whereas positively charged proteins will remain nearer to the cathode. The relative movement of each protein depends on its specific charge determined by its amino acid ...

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