1. Agarose Gels for IEF
1.1. Gel Preparation
a) Three agarose IEF gels (180 mm in length and 3.4 mm in diameter) containing 1 M thiourea and 6 M urea were prepared.
b) Agarose IEF (0.10 g) and D-sorbitol (1.2 g) were put into a 50-mL beaker, and dissolved in 5.8 mL distilled water at room temperature (solution A).
c) Solution A was boiled 10 times for 15 s in a microwave oven until the solution became clear, and was kept in a 70°C water bath for 5 min.
d) A mixture of urea (3.6 g) and thiourea (0.76 g) powder was put into solution A at 70°C, which we shall call solution B hereafter. The volume of solution B was then adjusted to 9.0 mL with distilled water, and was kept at room temperature.
e) Solution B was divided into three test tubes: 1.0 mL for acidic, 4.5 mL for neutral, and 2.5 mL for basic solutions.
f) Four kinds of Pharmalyte (pH 2.5–5.0 for acidic, pH 3.0–10.0 and pH 4.0–6.5 for neutral, and pH 8.0–10.5 for basic solutions) were added to the respective tubes according to the protocol shown in Table 1.
g) A glass tube (standard size: 260 mm × 3.4 mm ID) was prepared beforehand, the bottom of which was covered with a piece of dialysis membrane and tied with a rubber band.
h) The glass tube was attached to AE-6300 electrophoresis unit (apparatus for agarose IEF produced by ATTO).
i) The acidic, neutral, and basic solutions were sucked in with one syringe each through thin 30-cm-long polyethylene tubing. First, the acidic solution was gently injected into the glass tube until the meniscus reached a height of 20 mm from the bottom of the tube. Next, the neutral solution was slowly injected until the meniscus reached a height of 135 mm from the bottom, and then the basic solution was carefully injected until the meniscus was 180 mm from the bottom. A 10 μL overlaying solution was gently layered on top of the agarose solution, which made it easier for proteins to enter the agarose IEF gel. The glass tube was filled with the chamber and kept there at least 6 h, until the agarose solution gelled.
1.2. Sample Preparation
Freshly obtained mammalian tissues were cut into small blocks, quickly frozen in liquid nitrogen, and stored at –80°C until use. Each frozen tissue piece (about 10 mg) was homogenized with a Teflon glass homogenizer in extraction medium (20 times the volume of the tissue pieces) (e.g., brain, muscle, kidney, and liver). Homogenates of the tissues were centrifuged at 112,000g for 20 min, and the clear supernatant was subjected to the agarose IEF gel.
1.3. Sample Application
a) We added 10–200 μL of protein sample solution at the cathodic end of the gel, and gently filled the overlaying solution above the sample solution to the top of the glass tube. We added anode buffer to the lower reservoir, and cathode buffer to the upper reservoir. First-dimensional IEF was conducted at 600 V for 18 h at 4°C.
b) Then the agarose gel was transferred onto the top of the second-dimensional SDS gel, either directly or after proteins in the gel were fixed.
2. Second-Dimensional SDS-PAGE
a) Slab gels for second-dimensional electrophoresis were 12% polyacrylamide gels (200 mm × 120 mm × 1.5 mm). Second-dimensional SDS-PAGE was carried out according to the stacking system of Laemmli with the slight modification of adding 1% SDS both in the stacking and separation gels.
b) The first-dimensional agarose IEF gel was loaded on top of the stacking gel without SDS equilibration and covered with 1% agar solution to keep the agarose IEF gel in place. An incubation medium was overlaid on the IEF gels.
c) The second-dimensional gel electrophoresis was started with a constant current at 40 Ma for 1 h and continued at 70 mA until the end of the run.
d) The slab gels were first soaked and shaken overnight in a destaining solution, which removed Pharmalytes from the gel (see Note 6). The slab gels were then stained with a staining solution containing PhastGel Blue R and destained with the destaining solution.
3 Comparison of IPG-Dalt With Agarose 2-DE
a) In this study, we compared the agarose 2-DE system with IPG-Dalt (immobilized pH gradients for IEF in the first dimension, SDS-PAGE in the second dimension) to assess their capability of analyzing high-molecular-mass proteins.
b) The first-dimensional IEF with IPGs was performed essentially as described by Gorg et al. IPG dry strips (Immobiline DryStrips pH 3.0–10.0 L) and Multiphor II (Amersham Biosciences) were used in this study.
c) Coomassie-stained 2-DE patterns of rat duodenum (Fig. 1) revealed that high-molecularmass proteins larger than 150 kDa could be analyzed with the agarose 2-DE but not with IPG-Dalt.
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Reference
- Walker, J. M. (Ed.). (2005). The proteomics protocols handbook. Humana press.
