Protein separation by electrophoresis

Protein electrophoresis is the process of separating proteins by placing them in a gel matrix and then observing protein mobility in the presence of an electrical field. The most commonly used technique is sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). In these gels, protein mobility is a function of the protein’s length and charge. Because proteins are normally folded and the amino acids in the polypeptide chain have different charges, it is important to make all proteins in a mixture have the same charge per unit length and the same shape if you want to compare their sizes by PAGE. This uniform protein shape and charge proportional to size is achieved by adding SDS detergent to remove secondary and tertiary protein structures.

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The anionic SDS coats the proteins, mostly in proportion to their molecular weight, and confers the same negative electrical charge relative to size across all proteins in the sample. Glycosylated proteins may not migrate at their expected molecular weight because their migration is based more on the mass of their polypeptide chains, not the sugars that are attached (Sambrook J et al. (1989) Molecular Cloning: A Laboratory Manual, vol I. Ed 2. Cold Spring Harbor Laboratory Press).

The most widely used gel system for separating a broad range of proteins by SDS-PAGE is the Laemmli system (Nature 227:680 (1970)), which uses Tris-glycine gels comprising a stacking gel component that helps focus the proteins into sharp bands at the beginning of the electrophoretic run and the resolving gel, where higher gel percentages separate the proteins based on their size. This classic system uses a discontinuous buffer system where the pH and ionic strength of the buffer used for running the gel (Tris, pH 8.3) is different from the buffers used in the stacking gel (Tris, pH 6.8) and resolving gel (Tris, pH 8.8).


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The highly alkaline operating pH of the Laemmli system may cause band distortion, loss of resolution, or artifact bands.

The major causes of poor band resolution with the Laemmli system are:

  • Hydrolysis of polyacrylamide at the high gel-casting pH, resulting in a short shelf life of 8 weeks
  • Chemical alterations such as deamination and alkylation of proteins due to the high pH of the separating gel
  • Reoxidation of reduced disulfides from cysteine-containing proteins, as the redox state of the gel is not constant
  • Cleavage of Asp-Pro bonds of the proteins when heated at 100°C in the Laemmli sample buffer, pH 5.2



Novex precast protein gels: the beauty of a straight line


Novex precast protein gels: the beauty of a straight lineAs discussed earlier, the key use of protein gels is to separate proteins for subsequent transfer onto a membrane for interrogation with antibodies, a process called western blotting. The NuPAGE Transfer Buffer maintains neutral pH and prevents reoxidation of reduced samples during protein transfer to a membrane. This avoids sample modifications that can occur at the alkaline pH of traditional transfer buffers and maintains sample antigenicity. NuPAGE Bis-Tris gels are able to separate proteins using lower acrylamide concentrations than are required for Tris-glycine gels. This more open gel matrix allows for more efficient transfer of proteins to membranes during western blotting.


Figure 1. Novex NuPAGE SDS-PAGE Gel System compared with standard Laemmli system.

Integrity of samples is maintained throughout electrophoresis with the Novex NuPAGE SDS-PAGE Gel System (left), compared to samples prepared with Laemmli (Tris-glycine) sample buffer (right)