ISOELECTRIC

FOCUSINGM. KALEEM IQBAL

0309-MPHIL-BIO-T-12

INSTITUTE OF INDUSTRIAL BIOTECHNOLOGY

GCU, LAHORE.

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.

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.

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.

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.

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.

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.

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.

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.

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.

A Slab Gel System for IEF.

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.

Questions??

THANKS