About the technique of Isoelectric Focusing for separation of proteins, its types and applications.

1. ISOELECTRIC
FOCUSINGM. KALEEM IQBAL
0309-MPHIL-BIO-T-12
INSTITUTE OF INDUSTRIAL BIOTECHNOLOGY
GCU, LAHORE.

2. Isoelectric Focusing
 Process by which
amphoteric molecules can
be separated on the basis of
their isoelectric points.
 Basic principle involved is
electrophoresis.
 Proteins are subjected to
electric field in a pH
gradient.
 Requires a solid surface
normally Polyacrilamide.

3. Iso-electric Point
(pI)
 The pH at which net charge on protein
becomes zero.
 Below pI = Positive Charge.
 Above pI = Negative Charge.
 Proteins move toward the electrode
with the opposite charge.
 During motion, proteins will either pick
or loose protons.
 Different from conventional
electrophoresis migrate to steady state.

4. Ampholytes
 Establishment of stable pH
gradient is important.
 Achieved by means of
commercially available
synthetic carrier amphoteric
electrolytes.
 600 – 900 Da.
 Closely spaced pI and high
conductivity.
 The curve is determined by
pH interval covered by the
ampholyte and the distance
between electrodes.

5. Working Procedure.
 Following Chemicals are required:
 Acrylamide solution.
 Water.
 Ampholyte solution pH 3.5 – 10.
 Ampholyte solution pH 4 – 6.
 Urea.
 Spin gently to mix urea.
 Add 10% APS and TEMED at the end.
 Remove bubbles. Fill the cassette completely with
solution.
 Allow to polymerize at room temperature.

6. Set UP Gel
 Remove the comb carefully after
gel has polymerized.
 Attach gel to the electrophoresis
tank according the
manufacturer’s instructions.
 Add catholyte (sodium
hydroxide) to the upper buffer
chamber and anolyte
(Phosphoric acid) to the lower
buffer chamber.

7. Sample Preparation &
Loading
 Mix protein sample with equal volume of 2X loading buffer.
 Loading buffer includes the following reagents:
 Urea.
 Ampholyte solution pH 3.5 – 10.
 Ampholyte solution pH 4 – 6.
 Triton X-100.
 2-Mercaptoethanol.
 1% bromophenol blue.
 Distilled water.
 Centrifuge the sample to remove any aggregate.
 Apply the supernatant to the wells with a disposable tip or
Hamilton syringe.
 Run the gel at 150V for 30 min and then at 200V for 2 hours.

8. Cutting slices for pH
determination
 After electrophoresis, gel is cut into 0.5cm slices
keeping track of the distance from any electrode.
 Place each slide into an eppendorf and label by
the distance (in cm) from the anode.
 Incubate each slice in 1ml 10mM KCl for about 30
min.
 Centrifuge and read pH of the clear supernatant.
 A standard curve is obtained by plotting pH of the
gel slices and the distance from the anode.
 Unknown pI can be determined.

9. Fixing and Staining the
gel.
 Soak the gel in 10% TCA for the removal of ampholytes.
 Ampholyte removal is necessary to reduce background staining.
 Stain the gel with Coomassie blue for 10 min.
 Discard staining solution and replace with the destaining solution.

10. IEF in horizontal slab gels.
 Horizontal slab gel has several advantages.
 Cooling of the gel is efficient as the gel remains
flat on the cooling plate.
 Lesser amount of electrode solutions are required.
 Gel size can be adjusted.
 Sample can be added at any position on pH
gradient.
 Precast IEF gels are commercially available.

11. A Slab Gel System for IEF.

12. 2D Gel Electrophoresis.
 An application of IEF.
 Protein separated in two
dimensions.
 First on the base of pI.
 Can be performed in tubes
of small diameter.
 Second on the basis of mol.
Wt. in normal SDS PAGE.
 Procedure can be
adapted by combining IEF
and PAGE.

13. Questions??
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