Isoelectric Focusing Technique combining ideas of isoelectric points and electric fields Very high resolution technique for protein 1,3

pI Isoelectric focusing uses the theory of protein pI –pI is the pH at which a given protein has a neutral overall charge The pI is dependant on which type of residues are present and how many –Bases make proteins positive and acids negative pI is very specific for each protein 1

How to Isoelectrofocus Establish a pH gradient Establish a voltage Stain your macromolecule (usually protein) Go do something while proteins move 1

What Happens Protein is loaded at the top of a column where pH is very high –Most things are negatively charged at this pH –Protons are stripped from residue side chains Proteins move in the electric field toward the distant cathode and away from the nearby anode As the proteins move through the pH gradient, they gain positive charge and reach neutrality At pH=pI, the proteins have no charge and stop 1

What Happens Proteins stop exactly at pH=pI and the stained proteins are very visible 1,4

Making a Gradient Column filled with low- density gel to allow proteins to move Highly stable ampholytes are molecules with specific pKa to give a specific and unchanging pH gradient 1,2

Why Use IEF We don’t use it for protein purification, affinity chromatography has taken over that area Can be used 2D with PAGE to see if a certain protein is present in a sample May be evidence that cells use it to move proteins around, especially with phosphorylation of macromolecules (gives negative charge) 1

References 1.Voet, D. Voet, J. G. Pratt. C. W. Fundamentals of Biochemistry: Life at the Molecular Level. 3 rd edition. John Wiley and Sons. (2008) Pictures: a/NOTES/Protein_Properties/protein_purificati on.htm