| 查看: 520 | 回复: 1 | |||
夕凝夕凝金虫 (小有名气)
|
[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
|
|
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 ] |
回复此楼» 猜你喜欢
086003调剂求助
已经有22人回复
-
生物与医药调剂名额多，专业范围宽食品生物相关的07，08，09，10都可调
已经有15人回复
-
食品科学论文润色/翻译怎么收费?
已经有256人回复
-
淮北师范大学生物医药还有11个调剂名额和资源环境还有10调剂名额，欢迎大家赶紧调剂！
已经有0人回复
-
食物营养学 电子版
已经有0人回复
-
电子科技大学（深圳）高等研究院陈明豪课题组招收推免硕士研究生
已经有19人回复
-
金铜纳米合成
已经有0人回复
-
求助1977版中国药典，非常感谢！！！
已经有1人回复
-
萝卜干防腐 馊酸味 微生物
已经有0人回复
» 本主题相关价值贴推荐，对您同样有帮助:
- 请高手帮忙分析一下这个PCR产物凝胶电泳图
已经有5人回复
- 能否批量修改电泳胶图？？
已经有10人回复
- DGGE 垂直电泳 灌胶技术 各位大侠帮帮忙
已经有13人回复
- 同工酶电泳图片如何处理，请帮忙分析如下照片，谢谢
已经有9人回复
- 请大家帮忙看看我的蛋白电泳图，谢谢大家了
已经有10人回复
- 求大侠们帮忙翻译一段文字（汉语翻译成英语）
已经有5人回复
- 外文期刊中蛋白质电泳图（SDS-PAGE）如何处理
已经有4人回复
- 外文期刊要求图和文字分开是什么意思
已经有8人回复
夕凝夕凝
金虫 (小有名气)
- 应助: 3 (幼儿园)
- 金币: 1447.9
- 散金: 18
- 红花: 8
- 帖子: 205
- 在线: 58.4小时
- 虫号: 2327281
- 注册: 2013-03-07
- 性别: MM
- 专业: 食品科学
2楼2015-01-06 16:36:05
