Biology Reference
In-Depth Information
7. IEF is then carried out to a total of 55,100 Vh with the following
parameters: (a) 200 V, 200 Vh; (b) 500 V, 250 Vh; (c) 1,000 V,
500 Vh; (d) 1,000-8,000 V, 4,500 Vh; and (e) 8,000 V,
40,000 Vh. Voltage increases are on a stepwise basis for all
steps except for step (d) which is on a linear gradient mode.
IEF separation is conducted in the dark by covering the IPGphor
unit with the black cover provided with the unit. The tempera-
ture is maintained at 20°C.
8. The strips can be used for the second dimensional separation
immediately or they can be stored in capped glass tubes at −80°C
until use.
1. Prepare a 1-mm thick, 13% gel (18×20 cm) by mixing 12.35 mL
of 40% acrylamide/bis solution with 9.5 mL of 1.5 M Tris-
HCl pH 8.8 separating buffer, 380 mL of 10% SDS, 380 mL of
10% APS, 15.39 mL of water, and 19 mL of TEMED. Pour the
gel solution into the PROTEAN II xi gel-casting assembly,
leaving space for the insertion of the IPG strip (~0.5 cm from
the top of the short glass plate). Overlay with 500 mL of water-
saturated butanol. Allow the gel to polymerize for at least 6 h.
2. Equilibrate each of the IPG strips with 10 mL of equilibration
buffer containing 1% (w/v) DTT for 15 min on a rocker.
3. Discard the DTT-containing equilibration buffer and equili-
brate each of the strips with 10 mL of equilibration buffer
containing 2.5% (w/v) of IAA for 15 min.
4. Briefl y rinse the equilibrated strips with ultrapure water.
5. Place the IPG strip on the top of the 13% gel and seal with
0.75% agarose (see Note 12).
6. Separate the proteins in the second dimension using 15 mA/gel
for 20 min, followed by 30 mA/gel at 10°C until the bromophe-
nol blue dye front comes close to the bottom of the gel.
7. After electrophoresis, wrap the gels together with the glass
plates undetached in plastic wraps to keep them moist and keep
them in the dark.
3.8. Second-
Dimension SDS PAGE
3.9. Image Acquisition
and Analysis
1. Gels with glass plate intact are rinsed with ultrapure water and
wiped dry before placing onto the platen of the Typhoon 9410
scanner for image acquisition (see Note 13).
2. Optimal excitation/emission wavelengths for detection are
Cy2: 488/520 nm, Cy3: 532/580 nm, and Cy5: 633/680 nm,
respectively.
3. A preliminary scan at low resolution of 1,000 mm and the
Photomultiplier tube (PMT) voltage being set at 500 V on
each channel (Cy2, Cy3, and Cy5) are conducted to assess
the overall profi le of the 2D gel.
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