1 principle of 2-d electrophoresis 1. first dimension: denaturing isoelectric focusing separation...

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

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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

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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

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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

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Highly Abundant Proteins

•Standard Strip Holder

•Manifold

P araffin o il

P araffin o il

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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.

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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

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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

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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.

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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