Isoelectric Focusing

• Technique combining ideas of isoelectric points and electric fields

• Very high resolution technique for protein

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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 protein1

How to Isoelectrofocus

• Establish a pH gradient

• Establish a voltage

• Stain your macromolecule (usually protein)

• Go do something while proteins move

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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 stop1

What Happens

• Proteins stop exactly at pH=pI and the stained proteins are very visible

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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

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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)

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References

1. Voet, D. Voet, J. G. Pratt. C. W. Fundamentals of Biochemistry: Life at the Molecular Level. 3rd 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/

bioc462/462a/NOTES/Protein_Properties/protein_purification.htm