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夕凝夕凝金虫 (小有名气)
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[求助]
璇峰府蹇欐寚鍑鸿繖娈垫枃瀛楁秹�?婁簡閭ｄ簺鐢垫吵鎶�鏈紵
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SDS-PAGE was carried out as described by Laemmli on 12% polyacrylamide gels. For runs under nonreducing conditions, 2-mercaptoethanol was omitted in the denaturing buffer. Polypeptide bands were stained with Coomassie blue R-250. The Mr of the polypeptides was determined by comparison with a standard protein solution (GE Healthcare, Milan, Italy) containing phosphorylase b (94 kDa), bovine serum albumin (67 kDa), egg albumin (45 kDa), carbonic anhydrase (29 kDa), trypsin inhibitor (20 kDa), and lysozyme (14 kDa). Nonreducing/reducing double SDS-PAGE was performed as follows. After SDS-PAGE under nonreducing conditions, the lane of interest was excised with a razor blade and incubated in 3 mL of equilibration buffer (375 mM Tris-HCl, pH 8.8, 6 M urea, 2% SDS, 20% glycerol) with 65 mM DTT for 20 min and then with 245 mM iodoacetamide in the same buffer without DTT for 20 min. The gel lane was then placed horizontally on the top of a 15% polyacrylamide gel and sealed with 0.5% agarose, prepared in Laemmli running buffer. For two-dimensional IEF/SDS-PAGE, isoelectric focusing (IEF) was performed on 7 cm, pH 3-10 nonlinear IPG strips (GE Healthcare). The strips were rehydrated overnight in a solution containing 7 M urea, 2 M thiourea, 2% CHAPS, 65 mM DTT, and 2% IPG buffer pH 3-10 (GE Healthcare) containing 10 μg of the protein sample. Strips were focused at 8000 Vh, with a maximum of 2500 V, at 20 ℃ using a Multiphor II electrophoresis unit (GE Healthcare). Prior to the second dimension, strips were incubated in equilibration buffer with 65 mM DTT for 10 min and then with 245 mM iodoacetamide in the same buffer without DTT for 10 min. [ ????????? http://muchong.com/3g ] |
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2楼2015-01-06 16:36:05
