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z631535213木虫 (正式写手)
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[求助]
关于细胞裂解的一点问题 已有3人参与
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准备自己配点裂解液用用，看了些东西，发现一些裂解液中提到有效裂解成分，我目前只看到三个，Triton X-100，NP40，SDS。 想问下对这方面有了解的虫友 1.曲通，NP40，SDS一般多少浓度可有效裂解细胞。主要是用来裂解细胞提取DNA来进行后面的实验 2.曲通，NP40，SDS对细胞的裂解作用能具体讲一讲么。 恩，大概就这些。拜谢  |
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z631535213
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2楼2014-10-17 08:28:43
jurkat.1640
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3楼2014-10-17 08:58:00
z631535213
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4楼2014-10-17 10:31:23
5楼2014-10-22 21:34:59
6楼2014-10-23 10:03:54
【答案】应助回帖
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Detergents are amphipathic molecules, meaning they contain both a nonpolar "tail" having aliphatic or aromatic character and a polar "head". Ionic character of the polar head group forms the basis for broad classification of detergents; they may be ionic (charged, either anionic or cationic), nonionic (uncharged) or zwitterionic (having both positively and negatively charged groups but with a net charge of zero). Detergents can be denaturing or non-denaturing with respect to protein structure. Denaturing detergents can be anionic such as sodium dodecyl sulfate (SDS) or cationic such as ethyl trimethyl ammonium bromide. These detergents totally disrupt membranes and denature proteins by breaking protein:protein interaction. Non-denaturing detergents can be divided into nonionic detergents such as Triton* X-100, bile salts such as cholate and zwitterionic detergents such as CHAPS. A major factor determining the behavior and interaction of molecules in biological samples is their hydrophilicity or hydrophobicity. Most proteins and other molecules with charged or polar functional groups are soluble (or miscible) in water because they participate in the highly ordered, hydrogen-bonded intermolecular structure of water. Some other proteins (or at least parts of proteins), as well as fats and lipids, lack polar or charged functional groups; consequently, they are excluded from the ordered interaction of water with other polar molecules and tend to associate together in structures having minimal surface area contact with the polar environment. This association of nonpolar molecules in aqueous solutions is commonly called hydrophobic attraction. Generally, moderate concentrations of mild (i.e., nonionic) detergents compromise the integrity of cell membranes, thereby facilitating lysis of cells and extraction of soluble protein, often in native form. Using certain buffer conditions, various detergents effectively penetrate between the membrane bilayers at concentrations sufficient to form mixed micelles with isolated phospholipids and membrane proteins. Detergent monomers solubilize membrane proteins by partitioning into the membrane bilayer. With increasing amounts of detergents, membranes undergo various stages of solubilization. The initial stage is lysis or rupture of the membrane. At detergent:membrane lipid molar ratios of 0.1:1 through 1:1 the lipid bilayer usually remains intact but selective extraction of some membrane proteins occurs. Increasing the ratio to 2:1, solubilization of the membrane occurs resulting in mixed micelles. These include phospholipid-detergent micelles, detergent-protein micelles, and lipid-detergent-protein micelles. At a ratio of 10:1, all native membrane lipid:protein interactions are effectively exchanged for detergent:protein interactions |
7楼2014-10-23 10:18:14
z631535213
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8楼2014-10-23 12:43:54
