Activity gels for analysing DNA processing enzymes
Activity gels for analysing DNA processing enzymes
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用于分析 DNA 加工酶的活性凝胶
DOI：
10.1016/0168-9525(86)90181-2
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发表时间：
1986
影响因子：
11.4
通讯作者：
R. Izzo
中科院分区：
文献类型：
--
作者：
U. Bertazzoni;A. Scovassi;M. Mezzina;A. Sarasin;E. Franchi;R. Izzo
Acgd凝胶分析是研究DNA和RNA代谢以及与分子底物活性相关的蛋白质的一种强有力的和有前途的技术。它在筛选粗产物中的活性形式、寻找前体和降解特异性、进行比较性研究和寻找有前途的突变体等方面特别有用。巯基乙醇和EDT。Tin~-~也很重要，因为有些蛋白质在1-2小时内复性，而另一些需要18-24小时。每种酶都必须仔细地优化条件。关键的一步是快速去除SDS和亲脂性杂质（通常存在于商业SDS中），亲脂性杂质与蛋白质结合，往往会阻止再结晶。电泳后用异丙醇水溶液凝胶z.酶在SDS-PAGE中完全复性后的复性取决于几个因素，包括恢复天然多肽构象的内在能力、有利的环境和最佳复性条件。用粗匀浆得到的信号似乎比用纯化的~-S，SNME组分得到的信号强。酶为了解决这一问题，已经建议在凝胶中包埋纤维蛋白原或在将纯化的级分应用于凝胶之前添加变性蛋白质的异质混合物。在我们的手中，检测的效率显着增加，通过选择最佳等级的SDS和洗涤的凝胶，快速去除SDS后，胍在高摩尔浓度。这种处理可能是有用的，在解离非极性SDS杂质和有利于正确的蛋白质重折叠。还应考虑复性缓冲液的温度，因为当凝胶在0-4 ℃复性时，一些酶似乎产生更强的信号。酶活性通常通过使用放射性标记的前体或底物的测定来测量，尽管可以使用其他检测方法。整个过程中酶活性单位的回收率可以通过考虑凝胶上样样品的比酶活性和测定后活性带的放射性强度来估计。回收率通常在1 - 5%之间。事实上，为了在凝胶中获得可见信号，必须在测定中使用非常高比活度的放射性标记的前体或底物，从而将其浓度降低到Km范围以下。为了节省时间和减少使用时放射性化合物的消耗，活性凝胶技术已经小型化4，使得可以使用0.5-2 ml的酶反应混合物。一些技术提示是：（a）保持凝胶完整，永远不要让它干燥，如果必要的话，将它放在潮湿的萨兰包装上;（b）在玻璃烧杯中复性和洗涤凝胶，在温和的旋转运动下，使用抽吸来改变溶液;（c）在SDSPAGE后立即去除具有蛋白质标记物的泳道，用孔姆西蓝染色，以获得与活性凝胶相同长度的方式脱色67
Acgd~ gel ana/ysis is eme~ ng as a powerful and promising technique in the study of int~ lved in DNA and RNA metabolism as weH as ot~ r proteins whose activities are linked to mauumolecular substrates. It is parlicularly vak~ able in screening actit~ forms of e~,~ s in crude e~ acts, in searcMngfor pre~ ursor and degradation spe~, in amducgng comparagve s~ ural studies and in the search for prom'n mutants. mercaptoe~ anol and EDT.~ Tin~-~ is also important, since some proteins renature in 1-2 h while others need 18-24 h. Conditions must be carefully op" tunized for each enzyme. A crucial step is the rapid removal of SDS and of iipophilic contmninmlts (usually present in commercial SDS) which bind to proteins and tend to prevent renatu~ tiorL Some authors suggest washing detergent fro~,~! the gel with aqueous isopropanol after electrophoresis z. Refolding of enzymes after their complete d~-naturation during SDS-PAGE depends on several factors, including intrinsic capacity to regain native polypeptide conformation, favorable environment and optimal renaturing conditions. It appears that a stronger signal is obtained with a crude homogenate than with pmified fractions of the~-s, snme. en~ zyme. To obviate this, embedding fibrinogen in the geF or addition of a heterogeneous mixture of denatured proteins s before application of purified fractions to the gel, have been suggested. In our hands, the efficiency of detection is markedly increased by selecting the best grade of SDS and washing the gel, after rapid removal of SDS, with guanidine at high molar concentration. This treatment might be useful both in dissociating non-polar SDS impurities and in favoring correct refolding of the proteins. The temperature of the renstumtion buffer should also be considered, since some enzymes appear to produce stronger signals when gels are renatured at 0-4 C.Enzyme activity is generally measured by an assay using either radioactively-labeled precursors or substrate, although other methods of detection can be used. Recovery of enzyme activity units Irom the whole procedure can be estimated by taking into account the specific enzymatic activity of the sample loaded on the gel and the radioactive intensity of the actbre band after assay. Recovery is usually between I and 5%. In fact, to obtain visible signals in the gel it is necessary to u~ e very high specific activity of radioactively-la~ eled precursor or substrate in the assay and thus to lower its concentration below Km range. In order to save time and reduce consumption of radioactive compounds when used, the activity gel technique has been miniaturized 4 so that enzymatic reaction mixtures of 0.5-2 ml can be use& A few technical tips are:(a) keep the gel intact, never let it dry and, if necessary, nmnipulate it on moist Saran wrap; Co) renature and wash the gel in a glass beaker, under gentle rotary movement and use suction to change solutions;(c) remove the lane with protein markers just after SDSPAGE, stain it with Coonmssie blue, destain it in such a way that the same length as the activity gel is obtained 67
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DOI：
--
发表时间：
1983
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Tseng,BY;Ahlem,CN
通讯作者：
Ahlem,CN
来自酵母 DNA 聚合酶-引物酶复合物的 DNA 引物酶的多肽结构。
DOI：
--
发表时间：
1985
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Plevani,P;Foiani,M;Valsasnini,P;Badaracco,G;Cheriathundam,E;Chang,LM
通讯作者：
Chang,LM
小牛胸腺末端脱氧核苷酸转移酶的蛋白水解降解。
DOI：
--
发表时间：
1982
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Chang,LM;Plevani,P;Bollum,FJ
通讯作者：
Bollum,FJ
人脑脊液核糖核酸酶：检测相对于血清对应物的糖基化改变。
影响因子：
2.9
作者：
Schieven,GL;Blank,A;Dekker,CA
通讯作者：
Dekker,CA