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14. Load samples into the loading cups.
15. Cover samples with 3-4 drops of cover fl uid.
16. Close the lid and commence IEF. The following parameters
have been successfully used:
(a) Hold at 300 V for 3 h.
(b) Ramp to 1,000 V over 6 h.
(c) Ramp to 8,000 V over 3 h.
(d) Hold at 8,000 V for 8 h.
17. Remove strip gels from the ceramic manifold, and place them
in individual tubes.
Prepare equilibration buffer (see Note 15).
18. Dissolve 0.6 g DTT in 60 mL of equilibration buffer, and add
10 mL to each tube.
19. Incubate for 20 min at RT with gentle agitation.
20. Dissolve 1.5 g iodoacetamide in 60 mL equilibration buffer,
and add 10 mL to each tube.
21. Incubate for 20 min at RT with gentle agitation.
22. Proceed immediately with SDS-PAGE. Do not freeze equili-
brated strip gels.
Polyacrylamide gels must be cast at least 1 day prior to running
SDS-PAGE to allow polymerization to occur. Detailed step-by-
step instructions for gel casting can be found in the Ettan DALT II
system user manual and can be obtained from the GE Healthcare
website (
http://www.gelifesciences.com/aptrix/upp00919.nsf/
content/479E615AE5FE9261C1257628001CE63D?OpenDoc
ument&Path=Catalog&Hometitle=Catalog&entry=12&newrel&
LinkParent=C1256FC4003AED40-A905385FF0BEA0
FAC125701900490803_RelatedLinksNew-C21BEC677D8448
3.4. Polyacrylamide
Gel Casting and
SDS-PAGE
Table 1
DIGE experimental design for the analysis of secretome
changes during oncogenic-Ras/TGF-
b
-mediated EMT
Gel number
Cy3 (25
m
g)
Cy5 (25
m
g)
Cy2 (25
m
g)
1
MDCK (a)
21D1 (a)
Internal standard
2
21D1 + TGF-
β
(a)
MDCK (b)
Internal standard
3
21D1 (b)
21D1 + TGF-
β
(b)
Internal standard
4
MDCK (c)
21D1 (c)
Internal standard
5
21D1 + TGF-
β
(c)
MDCK (d)
Internal standard
6
21D1 (d)
21D1 + TGF-
β
(d)
Internal standard
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