SDS-PAGE method is used to separate protein prior to analysis by MS. And running buffers, such as glycine- or Tricine-based, will have an effect on the protein separation process. Tricine-based running buffer systems have been successfully applied in the separation of various types of proteins.

Although Tricine-dSDS-PAGE is a powerful tool in membrane protein separation, williams et al. (DOI:10.1002/elps.200500730) also explored other buffer systems in conjunction with the dSDS-PAGE approach and describe an orthogonal Bicine-dSDS-PAGE method for the analysis of membrane proteins from the anaerobic bacterium, Clostridium thermocellum. And they compared glycine-, Tricine-, and Bicine-dSDS-PAGE systems.

Firstly, thermocellum cell cultures were grown at 55 °C and harvested. And glycine-, Tricine-, and Bicine-dSDS-PAGE were used to separate the proteins in the first dimension using Laemmli protocol, then further separation in the second dimension. Due to SDS-PAGE gels and running buffers are normally operated in the 6.6-8.8 pH range, which makes Bicine-(pH range of 7.6-9.0) and Tricine-based (pH range of 7.4-8.8) buffer systems somewhat more suitable. And according to experimental results of the first dimension, the optimized parameters for gel separation in Bicine- and Tricine-dSDS-PAGE were obtained. Based on the optimized parameters obtained, large-format experiments were performed to further improve protein representation and increase protein loading capacity of the gel.

In the work of Williams et al., they found using electrophoretic buffer systems other than the traditional Laemmli approach increase resolution and representation of membrane proteins. And it was found that Bicine-dSDS-PAGE provided the best separation condition of all.

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