1. ISOELECTRIC FOCUSING
Electrophoretic method that separates proteins according to the iso-electric points
Is ideal for seperation of amphoteric substances
Seperation is achieved by applying a potential difference across a gel that contain a pH gradient
Isoelectric focusing requires solid support such as agarose gel and polyacrylamide gel

2. Isoelectric focusing gels contain synthetic buffers called ampholytes that smooth the pH gradients.
Ampholytes are complex mixtures of synthetic polyamino-polycarboxylic acids
Commercially available ampholytes are-
BIO-LYTE
PHARMALYTE

3. It gives good separation with a high resolution compared to any other method
Resolution depends on
The pH gradient,
The thickness of the gel
Time of electrophoresis,
The applied voltage,
Diffusion of the protein into the gel.

4. PREPARATION OF IEF GEL
Gel is polymerised
Second glass plate is placed on first
Mixture is poured over a glass plate which contain spacer
Carrier ampholytes (suitable pH) and riboflavin mixed with acrylamide solution

5. Ampholytes form a pH gradient between anode and cathode
Potential difference is applied
Electrode wicks are laid along the long length of each side of the gel
After the gel has set glass plates are prised apart
This takes 2-3 hr

6. Voltage is again applied for 30 min
Become stationary when they reaches isoelectric point
Proteins having positive charge will migrates towards the cathode. negatively charged protein will migrates towards anode
Voltage is again applied for 30 min
Samples applied by laying on gel filter paper soaked in the sample
The power is then turned off

8. Gel is stained with Coomasie Brilliant Blue
Destained
Gel is stained with Coomasie Brilliant Blue
This precipitaes the proteins and allows smaller ampholytes to be washed out
The gel is washed with trichloroacetic acid

9. A TYPICAL ISOELECTRIC FOCUSING GEL

10. Thank you