Biology Reference
In-Depth Information
By using the 2D fluorescence DIGE (Ettan DIGE) technique, three different
protein extracts are labeled with CyDye fluors (Cy2, Cy3, and Cy5, respec-
tively) that are subsequently separated on the same 2D gel (
see
Fig. 3
). Thus,
the process of detecting, identifying, and quantifying proteins is simplified.
Each of the three protein extracts is labeled with one CyDye. After labeling, the
three samples are mixed together and run on the same gel. In this way, the same
protein labeled with the different CyDye will migrate to the same position on
the 2D gel, which helps to limit errors due to experimental variation (
see
Fig. 4
and Color Plate 2, following p. 46). Each of the individual samples can be
visualized independently by selecting the appropriate excitation and emission
wavelength to scan for each CyDye (
see
Fig. 4
).
RN6390 (RsbU
+
) OD5
RN6390 (
rsbU
-
) OD5
RN6911(
Δ
agr rsbU
-
)OD5
Lip
Lip
1
Lip
Lip
1
Lip
2
Lip
2
Ge
h
Ge
h
Aly
Aly
Aly
1
Aly
1
Geh
Geh
Aly
Aly
Aly
1
Aly
1
Aly
2
Aly
2
Aly
2
Aly
2
Lip
Lip
1
Lip
Lip
1
Spa
Spa
Fhs
Fhs
Fhs
1
Fhs
1
Geh
F4
Geh
F4
Geh
F4
Geh
F4
Geh
F3
Geh
F3
Geh
F3
Geh
F3
Lip
F1
Lip
F2
Lip
F1
Lip
F2
Lip
F1
Lip
F1
Geh
F
Geh
F1
Geh
F
Geh
F1
Aur
Aur
Geh
F
Geh
F
Geh
F
1
Lip
F2
Lip
F2
Aur
SspB
SspB
SspB
1
SspB
1
SspB
SspB
SspA
SspA
SspB
1
SspB
1
SspA
SspA
Geh
F2
Geh
F2
SspB
2
SspB
2
Geh
F2
Geh
F2
SspA
1
SspA
3
SspA
1
SspA
3
SspB
2
SspB
2
SspA
1
SspA
3
SspA
1
SspA
3
Hlb
Hlb
F
Hlb
1
Hlb
1
SA1812
SA1812
LukD
LukD
SspA
2
SspA
4
SspA
2
SspA
4
SspA
2
SspA
4
SspA
2
SspA
4
Hlb
2
Hlb
2
Hla
Hla
LytM
LytM
HlgB
HlgB
1
GlpQ
1
GlpQ
1
GlpQ
1
GlpQ
1
Hla
3
Hla
3
HlgC
HlgC
SsaA
SsaA
SA0620
SA0620
SsaA
SsaA
IsaA
IsaA
SA0620
SA0620
SceD
SceD
IsaA
IsaA
SceD
SceD
SplA
SplA
Hla
1
Hla
1
Hla
2
Hla
2
SplA
SplA
GlpQ
GlpQ
SplB
SplB
SplB
SplB
GlpQ
GlpQ
Plc
Plc
IsaA
1
IsaA
1
Plc
Plc
IsaA
1
IsaA
1
SplA
1
SplA
1
SplA
1
SplA
1
SplF
SplE
SplF
SplE
SplF
SplE
SplF
SplE
SplC
SplC
IsaA
1
IsaA
1
SplC
SplC
IsaA
1
IsaA
1
SplF
2
SplF
2
SplF
2
SplF
2
SplF
1
SplF
1
SplF
1
SplF
1
SasD
SasD
SasD
SasD
SasD
1
SasD
1
SA2097
SA2097
IsaA
F
IsaA
F
SA2097
SA2097
SasD
2
SasD
2
EarH
EarH
SA2097
F
SA2097
F
SA2097
F
SA2097
F
RN6911
Δ
agr
RN6911
Δ
agr
RN6390
rsbU
+
SA0570
SA0570
EarH
EarH
RN6390
RN6390
RN6390
RN6390
Fig. 4. Difference gel electrophoresis (DIGE) labeling technique to illustrate differ-
ences in protein patterns of three protein extracts. Extracellular protein patterns of
wild-type RN6390 (blue), the isogenic
agr
mutant (green), and RN6390 comple-
mented with
rsbU
of
Staphylococcus aureus
COL (red). Prior to separation by 2D gel
electrophoresis technique, protein extracts of the respective strains were labeled with
CyDye fluorochromes [Cy2 RN6390, Cy3 RN6911, Cy5 RN6390 (RsbU
+
)]. A mixture
of 50 μg of each protein extract was separated on one gel. The proteins were detected
by using a Typhoon [Cy2: Blue2-Laser (488 nm), Cy3: Green-Laser (532 nm), Cy5:
the Red-Laser (633 nm)]. (
See
Color Plate 2, following p. 46.)
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