Isoelectric focusing (IEF) is an electrophoresis technique that separates proteins based on their isoelectric point (pI). IEF is performed in an immobilized pH gradient created by buffering acrylamide derivatives. During IEF, proteins will migrate through the pH gradient until they reach the point where they are electrically neutral and do not migrate further. This allows proteins to be resolved based on small differences in their pI. IEF has applications in clinical testing, research, and agriculture industries to analyze protein profiles in samples like blood, serum, muscle extracts, and seed extracts.

1. Isoelectric focusing
Introduction
Principle
Procedure
Applications
Reference

2. First dimension, Isoelectric focusing (IEF)

3. Isoelectric focusing
Electrophoresis in a pH gradient
Electrophoresis gradient
Separation method that resolves protein
markers on the basis of their isoelectric
points markers points
Successfully used in clinical, research, and
agricultural fields agricultural fields
Blood
Serum
Muscle Extracts
Seed Extracts

4. Cont…
- Ability to separate on total protein marker
profiles or enzyme protein marker
profiles
- Proteins with same molecular weights
will separate out by pH
- Testing time is quick (~2- 3 days)
- Ability to have permanent record of the
gel
- Pre cast gels available
- Versatile to a large number of crops for
the agriculture industry
- Seed or tissue extraction

5. W is IEF
hat
IEF is preformed in a pH gradient.
 Proteins are amphoteric molecules with acidic
and basic buffering groups (side chain).
 In basic environment, the acidic groups
become negatively charged.
 In acidic environment, the basic groups
become positively charged.
 The net charge of a protein is the sum of all
charges.
 Isoelectric point (pI): the pH where the charge
of a protein is zero.


6. The principle of IEF
The IEF is a very high resolution separation
method, and the pI of a protein can be measured.

8. 2-DE instruments, 1st dimension
Amersham Biosciences
Bio-Rad

9. Two ways to form pH gradient
A. Classic IEF technique
Carrier ampholyte generated pH
gradient.
B. modern IEF technique
Immobilized pH gradients

10. A. Carrier ampholyte generated pH gradient



First developed by Svensson, nature pH
gradient (1961).Svensson H. Acta Chem Scand
(1961) vol.15, p325.
Practical realization by Vesterberg (1969).
Artificial pH gradient: synthesis of a
heterogeneous mixtures of isomers of aliphatic
oligoamino-oligocarboxylic acid.

11. Synthetic carrier ampholyte v.s. natural
occurring ampholyte
 Synthetic carrier ampholyte:




High buffering capacity and solubility at the pI.
Good and regular electric conductivity at the pI.
Absence of biological effects.
Low molecular weight.
Natural occurring ampholyte:
 Amino acids or peptides
 Lack the properties above
 Can not be used in IEF.

12. Behavior of ampholytes

Negatively (-) charged carrier ampholyte
 Move toward anode (+).
 Such as -COOPositively (+) charged carrier ampholyte
 Move toward cathode (-).
 Such as NH3+

13. Solution in the IEF

To maintain a gradient as stable as possible,
electrode solutions are applied between the gel
and the electrodes.
 Acid is used at the anode.
 Base is used at the cathode.
–
Example: an acidic carrier ampholyte reach the
anode (+), its basic buffering group would protonated
(acquire a positive charge) from the medium and it
would be attracted back by the cathode.

14. Carrier ampholytes as solvents for proteins.

Carrier ampholytes also help to solublize
proteins, which stay in solution only in the
presence of buffering compounds.

They are necessary in traditional IEF and new
immobilized pH gradient IEF.

15. Problems for the traditional IEF
1. Long running time.


Protein close to their pI have low net charge thus have
low mobility.
Denatured polypeptides migrate slower in gel than
native protein.
2. Gradient drift.
 The pH gradient become instable during lone time
 Most basic proteins drift out of the gel.
3. Proteins behave like additional carrier ampholyte
 They modify the profile of pH gradient

16. B. Immobilized pH gradient, IPG
 First developed by Righetti ,(1990).
 Immobilized pH gradient generated
by buffering acrylamide derivatives
(Immobilines)
 Immobilines are weak acid or weak
H
base.

17. Advantage of IPG strips
1.
2.
3.
4.
5.
6.
7.
Industrial standard (GMP) reduce variation.
The chemistry of the immobiline is better
controllable.
The film-supported gel strips are easy to handle.
The fixed gradient are consistent during IEF.
Stable basic pH gradient allow reproducible
results for basic proteins.
High protein loads are achievable.
Less protein loss during equilibration in SDS
buffer.

18. Rehydration of IPG strip
 Standard rehydration solution:
8M urea, 0.5% CHAPS, 0.2% DTT, 0.5% carrier
ampholyte, 10% (v/v) glycerol, 0.002% bromophenol
blue
 Types of rehydration:
1.Rehydration cassette
2.Reswelling tray
3.Rehydration loading
4.Cup loading

19. 1. Rehydration cassette

Disadvantages:
1.
high volume of
rehydration solution
needs.
2.
cassette leaking due to
urea and detergent.
3.
rehydration loading of
different sample is not
possible.

20. 2. Reswelling tray

Rehydration volume must be controlled.




7cm: 125 mL
13 cm: 250 mL
18 cm: 340 mL
24 cm: 450 mL

21. Run IEF step 1
1. Remove protective film from Immobiline™ DryStrip gel.

22. Run IEF step 2
2 . Apply rehydration solution to the Strip Holder.

23. Run IEF 3
3. Wet entire length of IPG strip in rehydration solution by placing
IPG strip in strip holder (gel facing down).

24. Run IEF 4
4. Gently lay entire IPG strip in the strip holder, placing the end of
IPG strip over cathodic electrode.

25. Run IEF 5
5. Protein sample can be applied at sample application well
following the rehydration step if the protein sample was not included
in the rehydration solution.

26. Run IEF 6
6. Carefully apply DryStrip Cover Fluid along entire length of IPG
strip.

27. Run IEF 7
7. Place cover on strip holder.

28. Run IEF 8
8. Place assembled strip holder on Ettan™ IPGphor™ platform

29. Analysis of Protein Markers
Coomassie Blue Stain of
W
heat
Silver Stain of Soybeans

30. Silver Staining
Place the recently run gel into a staining
dish, pour 200 ml of fixative solution
onto the gel, rock for 15 minutes .
Wash the gel while rocking for one hour
in 500 ml of deionized water, repeat
wash step with fresh water.
Allow the gel to dry completely mite be
overnight at room temperature.
Clean all staining glassware and stir
bars with reducing wash solution.

31. Cont…..
Formulate the silver staining solution
Prepare gel for staining by washing for
5 minutes in 200 ml deionized water
minutes water
Pour silver stain mixture over the gel
Pour gel’’s surface and begin rocking
the gel.
Allow the gel to stain until bands reach
desired intensity

32. Cont….
Discard the staining solution and rinse
gel in deionized water gel water
Pour stop solution onto the gel, soak for
10 minutes.
Place the gel in 200 mL of deionized
water, soak for 10 minutes.
Allow gel to air dry

33. Points to be noted…
Thirty
minutes
before the
completion of the
gel run prepare the
enzyme stain
Pour the stain over
the gel surface
immediately after
focusing
rock and heat the
gel during staining
gel staining

34. Proteins components in the Thermo Scientific DyLight 549/649
Fluorescent MW Marker are dual-labeled with two fluorophores for
detection in two different fluorescent channels in gel (Panel 1) or on
membrane (Panel 2). The bands also can be detected with Coomassie
dye or silver stains (Panel 3).

35. Applications of Isoelectric Focusing
Agricultural Industry
1. Genetic Purity
2. Variety identification, including trait
3. Parental Line Maintenance Parental
Maintenance
4. Breeding Programs

36. References







Reiner Westermeier. Electrophoresis in Practice. 3 rd Edition.
WILEY-VCH, Weinheim (2001) page 3.
Coligan, J.E., et al. , Eds. (2002). Electrophoresis, In Current
Protocols in Protein Science, pp. 10.0.1-10.4.36. John Wiley
and Sons, Inc. New York.
Bollag, D.M., Rozycki, M.D. and Edelstein, S.J. (2002). Protein
Methods, 2nd ed. Wiley-Liss, Inc. New York.
Hames, B.D. and Rickwood, D. Eds. (1990) Gel Electrophoresis
of Proteins: a Practical Approach, 2nd ed. Oxford University
Press, New York.
Craig Nelson Manager Electrophoresis Technologies
BioDiagnostics Inc.
Diagram.Par.64776.Image.560.446.1.Integrating-IEFFractionato-jpg
2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim