【专题讨论】蛋白常用lysis buffer组成与作用详谈
丁香园论坛
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<strong><font>蛋白质技术中常常要用到lysis buffer，各个实验室的lysis buffer的配方是不同的，开设个专题，希望大家详细谈谈自己使用的lysis buffer的配方，以及各个组成成分的作用，方便广大蛋白质战友。</font></strong></center>
先介绍一下我们用的：
PBS
缓冲液，不用多说；
TRITON X-100
Triton X-100中文名为曲拉通X-100，分子式为t-Oct-C6H4-(OCH2CH2)xOH, x=9-10，平均分子量为647。 常用的非离子性去垢剂，主要是去垢的作用；
PMSF
PMSF即Phenylmethanesulfonyl fluoride，也称Phenylmethylsulfonyl fluoride或α-Toluenesulfonyl fluoride。中文名为苯甲基磺酰氟。分子式为C7H7FO2S，分子量为174.19。可抑制丝氨酸蛋白酶例如胰蛋白酶(trypsin)和胰凝乳蛋白酶(chymotrypsin)，也可抑制半胱氨酸蛋白酶和乙酰胆碱酯酶。有效浓度为0.1-1mM。难溶于水，且在水溶液中非常不稳定；
LEUPEPTINE
Inhibition spectrum: inhibits serine (trypsin (Ki=13 µM), plasmin, porcine kallikrein) and cysteine proteinases (papain, cathepsin B). Does not inhibit chymotrypsin and thrombin.
Mechanism of action: Competitive and reversible inhibitor. Inhbition may be relieved by an excess of substrate.
Properties: Soluble in water, ethanol, acetic acid and DMF (Stock solution: 10 mM)
MW: leupeptin: 426.6; leupeptin hemisulphate monohydrate: 542.7
Suggested final concentration: 1-10 µM (0.5-1 µg/ml)；
PEPSTAIN
Inhibition spectrum: potent inhibitor of various aspartic proteinases e.g cathepsin D, renin, pepsin, bacterial aspartic proteinases and HIV proteases. Not selective at all.
Mechanism of action: Transition state analog.
Properties: First isolated from culture filtrates of various species of Streptomyces. Analogs of pepstatin have been designed in order to increase the selectivity and potency of the inhibition. Soluble in DMSO or methanol (Stock solution: 1 mM); Stable at -20°C.
MW: 685.9
Suggested final concentration: 1-5 µM；
整个实验室都在用这个组成，蛋白沉淀加入lysis buffer之后，进行常规超声处理， 效果还可以。
先介绍一下我们用的：
PBS
缓冲液，不用多说；
TRITON X-100
Triton X-100中文名为曲拉通X-100，分子式为t-Oct-C6H4-(OCH2CH2)xOH, x=9-10，平均分子量为647。 常用的非离子性去垢剂，主要是去垢的作用；
PMSF
PMSF即Phenylmethanesulfonyl fluoride，也称Phenylmethylsulfonyl fluoride或α-Toluenesulfonyl fluoride。中文名为苯甲基磺酰氟。分子式为C7H7FO2S，分子量为174.19。可抑制丝氨酸蛋白酶例如胰蛋白酶(trypsin)和胰凝乳蛋白酶(chymotrypsin)，也可抑制半胱氨酸蛋白酶和乙酰胆碱酯酶。有效浓度为0.1-1mM。难溶于水，且在水溶液中非常不稳定；
LEUPEPTINE
Inhibition spectrum: inhibits serine (trypsin (Ki=13 µM), plasmin, porcine kallikrein) and cysteine proteinases (papain, cathepsin B). Does not inhibit chymotrypsin and thrombin.
Mechanism of action: Competitive and reversible inhibitor. Inhbition may be relieved by an excess of substrate.
Properties: Soluble in water, ethanol, acetic acid and DMF (Stock solution: 10 mM)
MW: leupeptin: 426.6; leupeptin hemisulphate monohydrate: 542.7
Suggested final concentration: 1-10 µM (0.5-1 µg/ml)；
PEPSTAIN
Inhibition spectrum: potent inhibitor of various aspartic proteinases e.g cathepsin D, renin, pepsin, bacterial aspartic proteinases and HIV proteases. Not selective at all.
Mechanism of action: Transition state analog.
Properties: First isolated from culture filtrates of various species of Streptomyces. Analogs of pepstatin have been designed in order to increase the selectivity and potency of the inhibition. Soluble in DMSO or methanol (Stock solution: 1 mM); Stable at -20°C.
MW: 685.9
Suggested final concentration: 1-5 µM；
整个实验室都在用这个组成，蛋白沉淀加入lysis buffer之后，进行常规超声处理， 效果还可以。
