Increasing the activity of affinity-purified DNA-binding proteins by adding high concentrations of nonspecific proteins.
Increasing the activity of affinity-purified DNA-binding proteins by adding high concentrations of nonspecific proteins.
复制标题
通过添加高浓度的非特异性蛋白质来提高亲和纯化的 DNA 结合蛋白的活性。
DOI：
10.1016/0003-2697(92)90353-9
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发表时间：
1992
影响因子：
2.9
通讯作者：
Ehrlich,M
中科院分区：
文献类型：
--
作者：
Zhang,XY;Asiedu,CK;Supakar,PC;Ehrlich,M
两个序列特异性的DNA结合蛋白的转录控制中牵连的活性大幅下降时，这些被亲和纯化，并在电泳迁移率变动测定的标准条件下进行测定。将反应混合物中牛血清白蛋白的浓度从0.1 mg/ml增加到5 mg/ml，刺激了这些亲和纯化的蛋白质、人CREB（环AMP反应元件结合蛋白）和MDBP（甲基化DNA结合蛋白）的DNA结合活性约5倍至20倍以上。在亲和纯化的MDBP的情况下，将亲和流通级分添加回测定混合物中，在低得多的最终蛋白质浓度下得到类似程度的刺激。通过向结合反应缓冲液中加入非离子去污剂，也增加了亲和纯化的CREB的特异性DNA结合活性，但没有MDBP的特异性DNA结合活性。在用亲和流过级分或5 mg/ml BSA补充亲和纯化的MDBP后观察到的MDBP·DNA复合物量的大幅增加表明是由于非特异性蛋白刺激特异性复合物的形成，而不是由于在测定期间防止吸附或变性导致的活性损失。
A large decrease in the activity of two sequence-specific DNA-binding proteins implicated in transcription control was seen when these were affinity purified and assayed under standard conditions in electrophoretic mobility shift assays. Increasing the concentration of bovine serum albumin in the reaction mixtures from 0.1 to 5 mg/ml stimulated the DNA-binding activity of these affinity-purified proteins, human CREB (cyclic AMP response element binding protein) and MDBP (methylated DNA-binding protein), approximately 5-to more than 20-fold. In the case of affinity-purified MDBP, adding back the affinity flow-through fraction to the assay mixture gave similar extents of stimulation at much lower final protein concentrations. The specific DNA-binding activity of the affinity-purified CREB, but not that of MDBP, was also increased by adding a nonionic detergent to the binding reaction buffer although not as much. The large increase in the amount of MDBP · DNA complex seen upon supplementation of the affinity-purified MDBP with the affinity flow-through fraction or 5 mg/ml of BSA was shown to be due to stimulation, by nonspecific proteins, of specific complex formation and not to prevention of activity losses by adsorption or denaturation during the assay.