Isoelectric Focusing

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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 lowdensity 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. 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/bioc462/4
62a/NOTES/Protein_Properties/protein_purificati
on.htm
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