1

Principle of 2-D Electrophoresis

• 1. First dimension:denaturing isoelectric focusingseparation according to the isoelectric point

• 2. Second dimension:SDS electrophoresisseparation according to the molecular weight

• 2-D electrophoresis resolves a few thousand protein spots

2 29-Apr-04

2-D electrophoresis: traditional method

sam ple

gel rod rebufferedin SDS buffer

Princip le according to P.H . O ’Farre ll (1975)

pH 10

pH 10

pH 3

pH 3

D enaturing isoelectricfocusing in presence

of urea, N onidet NP-40in vertica l ge l rod

First Dimension: Second Dimension:

SD S polyacrylam idegel e lectrophoresis

in d iscontinuous gradient gel

Separation acc. toIsoelectric Points (charge)

Separation acc. toM olecular W eight (m ass)

3

Add Sample to 1st Dimension Strips and Focus

4 April 28, 20044

Placing the Ettan™ SDS gel into the cassette

•

5 April 28, 20045

Place equilibrated IPG strip onto 2nd Dimension

acidic end

•gel surface up

6

Load and seal the IPG strip onto the gel surface

7

Insert Cassette into Ettan Dalt

8

Theoretical pI and Mr map of yeast cell proteins(calculated from MIPS data)

1

10

100

1000

2 4 6 8 10 12 14Theoretical pI

Mr

/ kD

a

From: Wildgruber et al. Electrophoresis. 21 (2000) 2610-2616.

9

Wide pH Gradient: 3 – 11 NL

•Mouse liver extract

•IPG 24 cm, pH 3-11NL

•From A. Görg

•Proteomics Department

•Technische Universität

•Munich

pH 3 pH 11

10 10

Why don`t we see this pattern?

•Post translational modifications

•Not all proteins are expressed

•Regulatory proteins are expressed in low copy numbers

•Missing proteins:

–hydrophobic

–high molecular weight

–very basic

•Proteome is not static!

11

The Dynamic Range of Expression: Avogadro´s Challenge

Copies / cell

10

100

1000

10,000

100,000

Fluor dye(1 ng)

20 mg

2 mg

200 µg

20 µg

2 µg

Coomassie (100 ng)

2,000 mg

200 mg

20 mg

2 mg

200 µg

12

2-D Electrophoresis of Mouse Liver Proteins

pH 4 pH 9

kDa

94

67

43

30

20

Görg et al.Electrophoresis16 (1996) 1079 - 1086

13 April 28, 200413

Wide pH gradient: 3 – 11 NL

•Mouse liver extract

•IPG 24 cm, pH 3-11

•From A. Görg

•Proteomics Department

•Technische Universität

•Munich

pH 3 pH 11

14

1491

1564

218 1429

Mouse liver proteins

From A. Görg et al. (1999)

IPG 4 - 5

IPG 4 - 7

IPG 5 - 6 IPG 5,5 - 6,7

Number of spots

15

IPG strips

with overlapping pIs

pH 3.0-5.6

pH 3-11

pH 6.2-7.5

pH 5.3-6.5

pH 7.0-11

16

Increased Resolution: Blow - Ups of Spots

IPG 4-7

IPG 5-6

IPG 4-7

IPG 5,5-6,7

Mouse liver proteins

From A. Görg et al. (1999)

17

Two-Dimensional Gel Based Proteomics

Low pI High pI1st Dimension IEF

Disease Tissue

High Mass

Low Mass

2nd Dim

ension SD

S-P

AG

E

123

4

5 6

8

7

39 9 1011

12

1314

15

16171819

20

2122

2324

25

26

27

2928

30

3138

3233 40

343736

35

Healthy Tissue

18

2-D Electrophoresis - Strengths

• Physico-chemical parameters of proteins measured

• Non-destructive separation of intact proteins

• Isoforms and post-translational modifications displayed

• Multiplexing, DIGE

• Quantitative method, internal standard (DIGE)

• High resolution, particularly after pre-fractionation

• High throughput, parallel runs

• Multiple detection, blotting, applicable

• Efficient fraction collector

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High Protein Loads•Problem: Highly Abundant Proteins

20 April 28, 200420

Highly Abundant Proteins

•Standard Strip Holder

•Manifold

P araffin o il

P araffin o il

21 April 28, 200421

Staining of IPG Strips (cont. urea, detergent)

• Acid Violet 17 Staining: (Patestos NP et al. Electrophoresis. 9 (1988) 488-496)

• fix for 20 min in 20% TCA,

• wash for 1 min in 3% phosphoric acid,

• stain for 10 min in 0.1 % Acid Violet 17 solution in 10% phosphoric acid,

• destain 3 in 3% phosphoric acid until background is clear,

• wash 3 1 min with H2Odist,

• impregnate with 5 % glycerol,

• air dry.

22 April 28, 200422

Casting SDS gels – important points

•HQ reagents: PlusOne labelled chemicals are a benchmark

•TEMED not too old

•Freshly made APS

•Precool monomer solution mix (containing the TEMED)

•Add APS short before use

•Pour solutions quickly in one go

23 April 28, 200423

EttanTM Dalt II Gel on film support•1 mg E. coli strain B

•IPGphor 24 cm pH 4 - 7

•ETTANTM Dalt gel 12.5 % T

•Colloidal CBBG250 staining

24

BACS-SDS gels

SynapticMembranePreparation

25

Blue Native PAGE

• for Membrane Protein Complexes

• Add Coomassie dye into cathodal tank

• Dye competes with nonionic detergents

• Negatively charged proteins (like SDS)

• No aggregation

• Soluble in detergent-free solution

• Proteins migrate as blue bands

Schägger, H.& von Jagow, G. (1991). Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form. Analytical Biochemistry 199(2), 223-31.

26

669 kDa

440 kDa

232 kDa

140 kDa

67 kDa

Marker Etioplast Chloroplast

2D Blue Native-PAGE/SDS-PAGE

Dr. L. Eichacker, Botanik, LMU München

Native Blue ElectrophoresisSeparation of complexes with PAGE in presence of Coomassie Brilliant Blue (no SDS)

Schägger H. In: Attardi GM, Chomyn A, Eds. Methods in Enzymology 264 (1996) 555-566.

Werhahn W, Braun H-P. Biochemical dissection of the mitochondrial proteome from Arabidopsis thaliana by three-dimensional gel electrophoresis. Electrophoresis 23 (2002) 640-646.

First Dimension

27

2-D Blue Native/SDS-PAGE

IA

IA/IB

IB

Ia3

Ia1ID;Ia2;Ia4

IEIFIL

IHIG

ICIKIIF

IIE

VCVB

VD

IIC

IIB

IIC

IIDIIA

VH

III2a

FBPVA

IIIb1;IIIb2 IIIb5;IIIb1;IIIb2

IIIb3Ia3Ia3 ?2

IIIbS

IIO

IIP

IIIb6

Chl

BN-PAGE (1. dimension)I/II I/II II/IV V III(3) III(1)

200.0

116.3

66.3

55.4

36.5

31.0

6.0

21.5

14.4

SDS-

PAG

E (

2. d

imen

sion

)

?1

Ia4

IVD

Chl

IVB

HP1

GGR

IVH

IVG

TL16

UP1

UC1

IIQ

IN,GIK

IIIb6

IVA

IIE/H

IIE

Second Dimension

28

Two-Dimensional Blue Native/SDS PAGE