Isoelectric focusing

Isoelectric focusing

1. IsoelectricFocusing (IEF)
AmandeepSingh
Assistant Professor,
Department of Biotechnology,
GSSDGS KhalsaCollege,
Patiala

2. Principle
•Isoelectricfocusingisusedfortheseparationof
amphotericsubstances.e.g.proteins
•Moleculesareseparatedaccordingtotheirisoelectric
points.
•Separationisachievedbyapplyingpotentialdifference
acrossthegelthatcontainsapHgradient.
•ThispHgradientisformedbyadditionofampholytes
intothegel.
•Ampholytesarecomplexmixtureofpolyamino
polycarboxylicacids.
•Acrylamide,agaroseorPolyethyleneglycol(PEG)
(4%)arecommonlyusedforpreparationofIEFgel.

3. Procedure
1.Preparation of IEF Gel
2.Addition of ampholytes
3.Generation of pH gradient
4.Sample application
5.Running of electrophoresis
6.Staining

4. +---+++-++--+++
-+-+---+-+-+-+--
--+-++-+----++++
++----+--+-+-+-+
----++++-+----++
-+-+--+-+-+++---+
+-----++--+-+--+-
+-+-+-+-+++-++
+-++-+++-+-+---
+---+-+-+----+-
++++++++-+-+-+-
-----+-+-+----++-
+---+---+-+---+-+-
+ + + + + + +
+ + + + + +
+ + + + +
+ + + +
+ + +
+ +
+
-
--
---
----
-----
------
-------
+ + + + + + +
+ + + + + + + +
+ + + + + + + +
+ + + +
+ + +
+ +
+
-
--
---
----
-----
------
-------
+
-
+ +
--
+ + +
---
+
Meet their IsoelectricPoint
Movement Stop
Preparation of IEF Gel Addition of ampholytes Generation of pH gradient Sample application
Zwittor
ion
(+)
(-)
Procedure
Polymerized
Acrylamide solution on a
glass plate
Randomly placed charges
of ampholytesin
acrylamide gel
On applying potential
difference, pH gradient is
established

5. 1. Preparation of IEF Gel
2. Addition of ampholytes
Carrier ampholytes+ Riboflavin
Acrylamide solution
Lower glass plate
SpacerSpacer
Carrier ampholytes& Riboflavin are
dissolved in acrylamide solution
Mixture is poured on a glassplatewith
attached spacers on sides

6. Lower glass plate
Upper glass plate
Bright Light
Photo-decomposition
Covered with upper glass plate
•Then these plates are illuminated by
bright light.
•Which causes photo decomposition
of riboflavin present in the gel.
•Which further produce free radicals.
•Free radicals thus produced causes
ploymerization/solidification of
acrylamide solution making it a Gel.
IEF gelUpper glass plate is moved apart

7. 3. Generation of pH gradient
Electrode wick
Electrode wick
Anode
(Phosphoric acid)
Cathode
(NaOH)
IEF gel thus prepared is then placed in
electrophoreticchamber
For making anode, a piece of filter paper (known as wick) is soaked in the solution of phosphoric acid and placed on one side of IEF gel.
For making cathode, a piece of filter paper (known as wick) is soaked in the solution of NaOHand placed on other side of IEF gel.
Electrophoretic
buffer

8. 4. Sample application
5. Running of electrophoresis
Apply Potential difference by turning ONthe power
Ampholytesform a pH gradient
Power is turned OFF
Sample is applied by laying on the gel, small
squares of filter paper soaked in the sample.
Power is turned ON
Sample (Protein) electrophoresis off the paper
into the IEF gel
Protein move according to their charge
towards their respective electrodes
Electrode wick
Electrode wick
Anode
(Phosphoric acid)
Cathode
(NaOH)
Electrophoretic
buffer

9. Proteins movement
Proteins below their
Isoelecticpoints (pI)
Proteins above their
Isoelecticpoints (pI)
Positively (+) charged Negatively (-) charged
Move toward cathode Move toward anode
As they move, they meet their
isoelectricpointby capturing
charges from ampholytes
present in the gel
As they move, they meet their
isoelectricpointby capturing
charges from ampholytes
present in the gel
Movement Stop
(become Zwittorion)
Movement Stop
(become Zwittorion)
•Different proteins stop at different places as all have different charges and all need equal opposite charges to
meet their isoelectricpoints (to become zwitterion).
•This causes the separation of protein molecules.

10. 6. Staining
•StainingisdonebyCoamassieBrilliantBlue
stain.Butitcannotbedonedirectlybecause
ampholyteswillstaintoo,givingatotallyblue
gel.
•Gelisfirstwashedwithfixingsolution(10%
tricholoroaceticacid)
•Thisprecipitatesproteininthegelandallow
muchsmallerampholytestobewashedout.
•Afterthat,gelisstainedwithCoamassie
BrilliantBlueandthendestained.