Protein mixtures can be separated by protein electrophoresis which exploits differences in the migration properties of different proteins in an electric field. One simple method is one-dimensional SDS - polyacrylamide gel electrophoresis. The method is based on the principle that by the addition of sodium dodecyl sulphate proteins are given the same charge per unit length. The protein-SDS complexes then separate in polyacrylamide gel electrophoresis according to their molecular size. 2D gel electrophoresis combines iso electric focussing, in which the proteins are first separated according to their iso electric points, with SDS polyacrylamide electrophoresis and this combination of techniques provides for extremely high resolution.

Step 1: Loading the protein mixture on the gel
Step 2: In the first dimension the proteins are separated according to their iso electric points
Step 3: Transfer of the IEF gel strip to an SDS gel
Step 4: In the second dimension the proteins are separated according to their mass

The proteins in the gel are detected either by labelling them prior to the separation (for example by radioactive labelling) or after separation using dyes such as Coomassie Brilliant Blue or the modern fluorescent reagents whose linearity over a large concentration range permits quantitation of the spots in the gel.

To identify the proteins by mass spectrometry the spots are cut out of the gel either manually or by using the spot picking robot.