Electrophoresis in a pH 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
- Ability to separate on total protein marker
profiles or enzyme protein marker
- Proteins with same molecular weights
will separate out by pH
- Testing time is quick (~2- 3 days)
- Ability to have permanent record of the
- Pre cast gels available
- Versatile to a large number of crops for
the agriculture industry
- Seed or tissue extraction
W is IEF
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
Isoelectric point (pI): the pH where the charge
of a protein is zero.
The principle of IEF
The IEF is a very high resolution separation
method, and the pI of a protein can be measured.
Two ways to form pH gradient
A. Classic IEF technique
Carrier ampholyte generated pH
B. modern IEF technique
Immobilized pH gradients
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
Synthetic carrier ampholyte v.s. natural
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.
Behavior of ampholytes
Negatively (-) charged carrier ampholyte
Move toward anode (+).
Such as -COOPositively (+) charged carrier ampholyte
Move toward cathode (-).
Such as NH3+
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.
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.
Problems for the traditional IEF
1. Long running time.
Protein close to their pI have low net charge thus have
Denatured polypeptides migrate slower in gel than
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
B. Immobilized pH gradient, IPG
First developed by Righetti ,(1990).
Immobilized pH gradient generated
by buffering acrylamide derivatives
Immobilines are weak acid or weak
Advantage of IPG strips
Industrial standard (GMP) reduce variation.
The chemistry of the immobiline is better
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
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
Types of rehydration:
1. Rehydration cassette
high volume of
cassette leaking due to
urea and detergent.
rehydration loading of
different sample is not
2. Reswelling tray
Rehydration volume must be controlled.
7cm: 125 mL
13 cm: 250 mL
18 cm: 340 mL
24 cm: 450 mL
Run IEF step 1
1. Remove protective film from Immobiline™ DryStrip gel.
Run IEF step 2
2 . Apply rehydration solution to the Strip Holder.
Run IEF 3
3. Wet entire length of IPG strip in rehydration solution by placing
IPG strip in strip holder (gel facing down).
Run IEF 4
4. Gently lay entire IPG strip in the strip holder, placing the end of
IPG strip over cathodic electrode.
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.
Run IEF 6
6. Carefully apply DryStrip Cover Fluid along entire length of IPG
Run IEF 8
8. Place assembled strip holder on Ettan™ IPGphor™ platform
Analysis of Protein Markers
Coomassie Blue Stain of
Silver Stain of Soybeans
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.
Formulate the silver staining solution
Prepare gel for staining by washing for
5 minutes in 200 ml deionized water
Pour silver stain mixture over the gel
Pour gel’’s surface and begin rocking
Allow the gel to stain until bands reach
Discard the staining solution and rinse
gel in deionized water gel water
Pour stop solution onto the gel, soak for
Place the gel in 200 mL of deionized
water, soak for 10 minutes.
Allow gel to air dry
Points to be noted…
completion of the
gel run prepare the
Pour the stain over
the gel surface
rock and heat the
gel during staining
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).
Applications of Isoelectric Focusing
1. Genetic Purity
2. Variety identification, including trait
3. Parental Line Maintenance Parental
4. Breeding Programs
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
2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim