Proteomics & Genomics Dr.
Vikash Kumar Dubey
Lecture 11: Isoelectric focusing (IEF)
We have studied in previous lectures that proteins have ionizable amino acids side chains.
Ionization state of these side chains and N-terminal amino group and C-terminal carboxyl
provides net charge to a protein at given pH. As pH changes ionization state of the amino
acids side chains also changes (can be described by the Henderson-Hasselbalch equation).
This results in change in overall charge on protein.
Proteins with different amino acid sequences have different amino acid side chains
composition. Thus, at a given pH net charge may be different. In other words, the
charge on protein depends on what kind of amino acids it has.
At acidic pH most proteins are positively charged while in alkaline pH negatively charged.
However, in between pH net charge on protein can vary considerably. This variation This
variation is because of different protein sequence or difference in post-translational
modification. pH where net charge on a given protein is zero, called isolectric point of the
protein. Isoelectric point is an important biophysical parameter.
If we do an electrophoresis in pH gradient (We shall also discuss how to make pH gradient
during later part of the lecture) and protein sample (protein mixture) is loaded at pH 7.0:
All proteins with isoelectric point above pH 7.0 will have positive charge and move towards
negative electrode. As they move towards negative electrode due to pH increase (due to pH
gradient) net positive charge on protein would decrease. When protein reaches pH which
equals isolectric point of protein, net charge on the protein becomes zero and electrophoretic
mobility becomes nil (protein remains confined near isoelectric point in the pH gradient.
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Proteomics & Genomics Dr. Vikash Kumar Dubey
All proteins with isoelectric point below 7.0 will have negative charge and move towards
positive electrode. As they move towards positive electrode there is a decrease in the net
negative charge on the protein due to pH decrease (due to pH gradient). When protein reaches
pH which equals isolectric point of protein, net charge on the protein becomes zero and
electrophoretic mobility becomes nil (protein remains confined near isoelectric point in the
pH gradient). Please refer to Fig. 1 for detail. This process of separating proteins based on
isoelectric point is called isoelectric focusing.
Figure 1: Process of separating proteins based on isoelectric point
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Proteomics & Genomics Dr. Vikash Kumar Dubey
How pH gradient is generated?
It is technically difficult to generate a stable pH gradient by buffer system. A stable pH
gradient is generated using Ampholytes. Ampholytes are synthetic, low molecular mass
heteropolymer of oligoamino and oligocarboxylic acids (containing both acidic and basic
groups). Using various combination of amino and carboxylic acid group, ampholytes with
different isolectric points are synthesized. Ampholytes have another property which makes
them very useful. They have very high buffering capacity at their isolectric point. Mixture of
ampholytes with different isoelectric point ranges are commercially available (pH 7-12; pH
4.0-7.0 etc). General structure of ampholytes are shown in Fig. 2.
Figure 2: General structure of ampholytes.
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Proteomics & Genomics Dr. Vikash Kumar Dubey
-Ampholytes of a given range (for example pH 2.0-12.0) is mixed with arcylamide/bis
acrylamide while being polymerized (no SDS). If this polymerization is done at pH 8.8 and
electrophoresis is performed, what happens?
Ampholyte molecules with maximum isolectric point (above pH 8.8) will have maximum
positive charge and move fastest and reach closest towards negative electrode. The ampholyte
with second maximum isolectric point will not move further because the space taken by
ampholytes with higher isolectric point. As ampholytes have high buffering capacity at
isolectric point a higher pH is maintained. As soon as it moves forward, it will experience a
pH higher than its isoelectric point. Protein will get negative charge and move back (Fig. 3)
Figure 3: pH gradient using ampholytes
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Proteomics & Genomics Dr. Vikash Kumar Dubey
Similar, ampholytes molecule with minimum isoelectric point will have maximum negative
charge (at pH 8.8) and move fastest and reach closest towards positive electrode. The
ampholytes with second minimum isoelectric point will not move further because the space
taken by ampholytes with lower isolectric point. As ampholytes has high buffering capacity
at isoelectric point a lower pH is maintained. As soon as it moves forward, it will experience a
pH lower than its isoelectric point. Ampholytes will get positive charge and move back. This
way a isoelectric point gradient is generated in solid support (like polyacrylamide). As
ampholytes have high buffering capacity at isolectric point, the isoelectric point gradient
effectively makes a pH gradient. Now refer to our discussion earlier in this lecture about
protein in pH gradient. What happens if we use pH gradient generated by ampholytes? Protein
will get separated according to isolectric point. As the pH gradient is in solid support,
diffusion is minimized after electric field is stopped. Gel is sliced in small pieces and proteins
with different isoelectic points are separated (Fig. 4)
Figure 4: Separation of proteins in pH gradient
As shown in Fig. 4, each cm of gel contains proteins having isoelectric point differing in one
unit. This may accommodate more protein per slice and effective purification may not be
achieved in a protein mixture having many proteins.
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Proteomics & Genomics Dr. Vikash Kumar Dubey
By carefully choosing range of ampholytes better separation may be achieved. For example
if we know the isolectric point of desired protein, range of ampholytes can be narrowed. If we
consider that the desired protein’s isoelectric point is 5.0, we may use ampholytes of range 4-
7 (3 unit pH difference) in 10 cm gel. This will result in 1cm slice accommodating proteins
differing in 0.3unit (10cm/3unit pH difference) difference in isoelectric point.
Other less common method for generating pH gradient
Some times immobilines are also used to generate pH gradient. Immobilines are derivatives of
acrylamide. It could be acidic or basic and polymerized (as we have seen in case of
acrylamide). Solutions of acidic and basic immobilines are used to generate pH gradient as
shown in Fig. 5. Once pH gradient is generated, the gel can be used for isoelectric focusing.
Figure 5: Preparation of pH gradient using immobilines
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