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