Isoelectric Focusing Technique combining ideas of isoelectric points

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

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

How to Isoelectrofocus • • Establish a p. H 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 p. H is very high – Most things are negatively charged at this p. H – 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 p. H gradient, they gain positive charge and reach neutrality • At p. H=p. I, the proteins have no charge and stop 1

What Happens • Proteins stop exactly at p. H=p. I and the stained proteins are very visible 1, 4

Making a Gradient • Column filled with lowdensity gel to allow proteins to move • Highly stable ampholytes are molecules with specific p. Ka to give a specific and unchanging p. H gradient 1, 2

Why Use IEF • We don’t use it for protein purification, affinity chromatography has taken over that area • Can be used 2 D 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: 2. http: //www. science-tube. com/ 3. http: //www. zeitnews. org/ 4. http: //www. biochem. arizona. edu/classes/bioc 462/4 62 a/NOTES/Protein_Properties/protein_purificati on. htm