Procedure
To assemble the polymerisation cassette wet the plain glass plate (size 260 x 200 mm2) with a few drops of water. Place the Gelbond PAGfilm, hydrophilic side upwards, on the wetted surface of the plain glass plate. The GelBond PAGfilm should overlap the upper edge of the glass plate for 1-2 mm to facilitate filling of the cassette. Expel ecxess water with a roller. Place the glass plate which bears the U-frame (0.5 mm thick) on top of the GelBond PAGfilm and clamp the cassette together. Put it in the refrigerator for 30 min.
Assembly of the gel casting cassette 
(Left): Assembly of the polymerisation cassette for IPG and SDS gel casting on plastic backing (Glass plates, GelBond PAGfilm, U-frame 0.5 mm thick)
(Right): Application of the GelBondPAGfilm onto the glass plate
IPG gel casting 
(1) Gradient mixer and connecting valve; (2) outlet tubing valve; (3) gel casting cassette
The acidic, dense solution is pipetted into the mixing chamber and the basic, light solution into the reservoir of the gradient mixer, an extra portion of the dense solution is prepared and pipetted into the mold prior to pouring the gradient.
After pouring the gradient into the precooled mold (refrigerator), the mold is kept at room temperature for 15 min to allow adequate levelling of the density gradient prior to polymerization for one hour at 50°C. After polymerization, the mold is kept at room temperature for at least 15 min. Then the IPG gel is removed from the mold and extensively washed with deionized water, impregnated with 2% glycerol, and dried at room temperature in a dust-free cabinet and, if not used immediately, covered with a plastic film for storage at -20°C. The dried gels can be stored frozen for at least one year.
Note: In order to ensure the reproducibility of the IPG gradient, the volume of the cassette should be constant. Therefore, it is recommended to check the volume of the cassette from time to time since it diminishes on ageing of the U-frame.
Note: When one of the chambers is emptying faster than the other, the resulting pH gradient will not be linear. Check if there is an air bubble in the connecting line or whether the speed of the magnetic stirrer is not appropriate!
1. 样品制备
样品裂解液(lysis solution):
8M urea, 4% CHAPS, 40 mM Tris base, 65 mMDTE
制备后的裂解液分装冻存－20℃备用。
如果必要，urea的浓度可增加到9或9.8M。
Triton X-100, NP-40及其他的非离子型去污剂或Zwitterionic detergents可取代CHAPS
对于脂蛋白，膜蛋白的分析，可以用如下的蛋白质裂解液：
7M urea, 2M thiourea, 4% CHAPS, 40 mM Tris base, 65 mMDTE
Presulubilization of protein in (boiling) SDS buffer, followed by dilution with urea lysis buffer.
Initially solubilized in 0.5-1% SDS,followed by dilution with at least an eight excess of 2-4% (w/v) NP40, Triton X-100, or CHAPS to reduce the final concentration to <0.25%. This dilution displaces the SDS from the proteins and replaces it with a nonionic or zwitterionic detergent.
制备2DE样品时常见的污染物
盐、小离子性分子(small ionic molecules)、离子型去污剂(ionic detergent)、核酸、多糖、脂类以及酚类化合物等，这些物质会直接影响2DE的效果。
