The Protein Man's Blog | A Discussion of Protein Research

Tris-Tricine SDS-PAGE (polyacrylamide gel electrophoresis) is used to separate protein / peptides ranging from 1-100 kDa molecular weights. This method varies from Laemmli SDS-PAGE by replacing Glycine pK (9.6) with Tricine (pK 8.15). Due to the variation in pK the resolution of high or low molecular weight proteins by both methods vary. Lower acrylamide concentrations of Tricine gels help in easy transfer of hydrophobic proteins during Western blotting. Tricine gels are suitable in isolating hydrophobic proteins from 2D gel for mass spectrophotometric analysis. It is also helpful to isolate membrane protein complexes from biological membranes. When urea is added in stacking gel it can easily separate two different proteins of the same molecular weight. Laemmli SDS-PAGE gel can separate high molecular weight proteins (20-200 kDa) but proteins less than 20 kDa are not clearly separated and diffused even if higher acrylamide concentration (4-20% gradient gels) are used. In contrast, Tricine SDS-PAGE gels can be used to separate proteins below 100 kDa only and especially 20 kDa or lower molecular weight proteins or peptides are very well separated by this method. Staining of Tricine gels is crucial as there is every chance of losing low molecular weight proteins from the gel during staining and destaining process. Fixing, Staining and Destaining should be rapid to avoid blotting out of these small proteins. 

Why outsource manufacturing?

It seems strange that companies will invest time, money, and effort into developing products to then outsource them to a third-party manufacturer, so why is there growth in companies like G-Biosciences, who offer OEM and custom manufacturing in B2B relationships?

cGMP, or current Good Manufacturing Practices, are regulations enforced by the US Food and Drug Administration (FDA) to ensure that systems are in place that assure proper design, monitoring, and control of manufacturing processes and facilities.

With the advancement of biochemical tools, studies on membrane proteins have grown substantially. Since membrane proteins are difficult to extract and purify, it requires optimization and an orchestrated execution. As described in our basics of membrane protein isolation blog, surface active agents are crucial for the manipulation of membrane proteins. The amphiphillic properties of these components promote the interaction of the membrane proteins, which are wrapped around by hydrophobic lipid bilayer in their native state, to become water soluble. Nonetheless, the solubility cannot be extrapolated into stability and restoration of the native functional structure; therefore, it is not necessary that a detergent (surfactant or a surface-active agent) can yield a suitable stable membrane protein fraction despite good extraction. Also, a detergent that has shown good results previously with a particular membrane protein might not work well with other membrane proteins. In the absence of gold standards or thumb rules for membrane protein extraction, it becomes imperative to understand the physiochemical characteristics of different detergents before extracting the proteins for example, the charge and degree of hydrophobicity of a specific detergent can allow a prediction of its behavior in a solution and interaction with the protein of choice.