The Proteomic Workflow

Before diving into our specific lab projects, let’s take a look at the proteomics workflow and follow the steps that we use to investigate the proteome. 

Organisms that have undergone experimental treatment, e.g. heat stress, are first prepared for protein analysis.  Two methods are commonly used to separate proteins for further analysis: two-dimensional gel electrophoresis (2D GE) and liquid chromatography.  Currently we are employing 2D GE. In the first dimension proteins are separated according to their charge, which depends on the pH environment of the gel.  Proteins are applied to a narrow but long (7 to 24 cm) strip with an immobilized pH gradient (IPG strips) that typically ranges from pH 4 to 7 (many variations thereof are possible).  When we apply a voltage to the strip proteins start to migrate according to their pH at initiation until they reach the pH at which their charge is zero (isoelectric point or pI), a process that is called isoelectric focusing (Fig. 1).

Figure 1: Isoelectric focusing is based on proteins having different charges in different pH environments.  Once proteins reach their isolectric point (pI) they stop migrating in an electric field (or focus).  Thus this method separates proteins according to their electric charge.