ReisingerEichacker - PowerPoint PPT Presentation

Title: ReisingerEichacker


1
How to analyze protein complexes by 2D
BN/SDS-PAGE
Veronika Reisinger and Lutz A. Eichacker Departmen
t for Biology I, Menzingerstr. 67, 80638 München
2
Workflow
Cell fractionation
Membrane isolation
Solubilization of protein complexes
BN-PAGE
Denaturation of protein complexes
SDS-PAGE
3
Detergents
Dodecyl-ß-D-maltoside
Digitonin
C56H92O29, Mr 1229.31 g/mol
C24H46O11 , Mr 510.62 g/mol
4
Membrane solubilization by Digitonin ( ) and DM
( )
H
H
Pet
H
M
Pet
H
M
Pet
H
Pet
H
E
E
Psa
Psa
10kDa
10kDa
psa
C
Psa
Psa
G
Psa
D
psa
C
Psa
Psa
G
Psa
D
Psa
Psa
Pet
F
Pet
F
Pet
F
Pet
F
PsbR
PsbR
17,5 kDa
17,5 kDa
17,5 kDa
psa
A
psa
B
psa
A
psa
B
J
1
I
J
1
I
D
L
B
G
D
L
B
G
K
K
psa
L
psa
psa
L
psa
binding
binding
binding
pet
pet
pet
pet
pet
pet
pet
pet
Psa
Psa
Psa
Psa
LhcA4
LhcA2
LhcA1
LhcA3
LhcA4
LhcA2
LhcA1
LhcA3
A
A
-
-
-
FNR
FNR
FNR
Psa
F
Psa
F
Psa
N
Psa
N
Pet
C
Pet
C
pet
A
pet
A
Pet
E
Pet
E
Pet
E
Pet
E
Below CMC, maintenance of membrane
Above CMC, solubilization
5
Effect of detergent concentration on membrane
solubilization
Decreasing molecular mass of protein complexes
1.1 mM ß-DM or 4.5 mM Digitonin
Increasing concentration of detergent micelles
6
Optimization of complex solubilzation
A
B
Digitonin (mM)
ß-DM (mM)
2.2
1.1
4.5
9.0
18
36
72
144
2.2
1.1
4.5
9.0
18
36
72
144
kDa
kDa
669
669
440
440
232
232
Unstained protein complexes
140
140
67
67
detergent concentration
detergent concentration
7
Optimized solubilization resolved by2D
BN/SDS-PAGE
8
Resolution analysis of protein complexes after2D
BN/SDS-PAGE
Schematic complex composition of the thylakoid
membrane
9
Use of different detection methods after BN-PAGE
or BN/SDS-PAGE approaches
10
Detection of protein complexes after BN-PAGE
11
Methods for the detection of proteins after 2D
BN/SDS-PAGE
12
In-gel staining
13
In-gel Coomassie staining of second dimension gels
14
Comparison of different staining methods after
BN/SDS-PAGE one gel, three stains
15
Methods for the detection of proteins2D
BN/SDS-DIGE
BN-PAGE
radioactive labelling
0min
SDS-PAGE
20 min
80 min
16
CyDye labeling of membrane protein complexes
Em
Ex
488 nm
532 nm
633 nm
17
2D BN/SDS-DIGE of plastid membrane complexes


18
Methods for the detection of proteins after 2D
BN/SDS-PAGE Immuno detection
BN-PAGE
Radioactive labelling
0min
SDS-PAGE
20 min
80 min
19
Detection of proteins in the native and
denatured state
No signal
Not all proteins are detected by antibodies in
the native state after BN-PAGE
Good signal
But in the denatured state after 2D BN/SDS-PAGE
the same proteins are well detected
20
Detection of protein assembly kinetics by
antibody detection after 2D BN/SDS-PAGE
BN-PAGE
timepoint
0
1
SDS-PAGE
2
3
Protein complexes
21
Methods for the detection of proteins after 2D
BN/SDS-PAGE radioactive labelling
BN-PAGE
radioactive labelling
0 min
SDS-PAGE
20 min
80 min
22
Analysis of assembly by 2D BN/SDS-PAGE Kinetic
of protein complex formation followed by
pulse/chase radiolabeling of de novo expressed
proteins
BN-PAGE
SDS-PAGE
Protein complexes
23
Summary forThe assembly analysis of protein
complexesafter 2D BN/SDS-PAGEProtein subunits
are identified by antibodies and/or mass
spectrometry,andare labeled by fluorescent dyes
and/or radioisotopes. Assembly is concluded
from the position of protein subunits in the 2D
-gelA change of the horizontal position of a
protein subunit from low to high molecular mass
reflects the progression of subunit assembly into
the corresponding protein complex
24
Step-wise assembly of proteins to complexes after
2D BN/SDS-PAGE Protein subunits reveal the
molecular mass increase of the complex
1. Native PAGE
2. Denaturing PAGE