Isoelectric focusing

IntroductionPrincipleProcedure ApplicationsReference

First dimension, Isoelectric focusing (IEF)

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

BloodSerumMuscle ExtractsSeed Extracts

Cont…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

What 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 charges. 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.

2-DE instruments, 1st dimension

Amersham Biosciences Bio-Rad

Two ways to form pH gradient

A. Classic IEF technique Carrier ampholyte generated pH

gradient.

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 oligoamino-oligocarboxylic acid.

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.

Behavior of ampholytes

Negatively (-) charged carrier ampholyte Move toward anode (+). Such as -COO-

Positively (+) 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 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

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 base.

H

Advantage of IPG strips

1. Industrial standard (GMP) reduce variation.2. The chemistry of the immobiline is better

controllable.3. The film-supported gel strips are easy to

handle.4. The fixed gradient are consistent during

IEF.5. Stable basic pH gradient allow

reproducible results for basic proteins.6. High protein loads are achievable.7. Less protein loss during equilibration in

SDS buffer.

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 cassette2.Reswelling tray3.Rehydration loading4.Cup loading

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.

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 strip.

Run IEF 7

7. Place cover on strip holder.

Run IEF 8

8. Place assembled strip holder on Ettan™ IPGphor™ platform

Analysis of Protein Analysis of Protein Markers Markers

Coomassie Blue Stain of Wheat

Silver Stain of Soybeans

Silver Staining 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.

Cont…..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

Cont….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

Points to be noted…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

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

Applications of Isoelectric Applications of Isoelectric Focusing Focusing

Agricultural Industry 1. Genetic Purity 2. Variety identification, including

trait3. Parental Line Maintenance

Parental Maintenance4. Breeding Programs

ReferencesReferences Reiner Westermeier. Electrophoresis in Practice.

3rd 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.

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