Isoelectricfocusing Isoelectric focusing is the separation of proteins based on their pIs. In 2D gels, the most common first dimension separation is done by isoelectric focusing. Protein mixture separated in step 1 is solubilized in an IEF buffer which contains denaturing agents, detergents, reducing agents, etc. Different buffers (T. Rabilloud Electrophoresis 1996, 17(5), 813-829) and treatments (vortex and sonic bath) are used to solubilize different types of proteins. Solubilized protein mixture is applied to IPG strip (acrlyamide gel coated onto a 0.5 cm wide plastic strip) and allowed to absorb into the gel matrix for 10 to 12 hours, referred to as rehyrdration. Then the strip is placed under constant current for several hours to focus the proteins. Several commercial IPG strips are available: 7cm , 11cm 17cm and 24cm are the most common lengths which determine the amount of protein mixture that can be separated. The complex protein mixture must be completely soluble in the IEF buffer under the electrophoresis conditions. Mass range of the proteins commonly separated by IPG strips is 5–100 kD. Salts, polysaccharides and charged organic molecules could interfere with IEF.  | |