Adenosine triphosphate (ATP) is the most abundant and primary carrier of the required energy for various functions in cells. Prolonged ischemia, reperfusion, anaerobic metabolism and lactate accumulation can lead to a dramatic decrease of ATP, cell swelling, cell rupture, and finally cell death by necrotic, necroptotic, apoptotic, and autophagic mechanisms. Due to drastic hydrolysis of ATP in vivo by ectoenzymes and poor cellular penetration, the direct delivery of ATP to the ischemic tissues is difficult.
To increase delivery of ATP to the tissues and protect from enzymatic degradation, encapsulation in liposomes has been proposed and demonstrated in various models of ischemia [1,2]. Studies on myocardial [1,3,4], liver [5-8], retina [9] and wound healing [10-12] ischemia have shown the ability of liposomal encapsulated ATP to prevent cell death and tissue dysfunction following ischemic events.
The encapsulation of ATP in liposomes markedly promotes its effectiveness by preventing the hydrolysis by extracellular enzymes, increasing ATP circulation time and enhancing its intracellular penetration. Depending on the type of the cell line and the target organ various types of liposomes with different surface charges such as anionic, cationic and neutral has been studied by various groups. Moreover, ATP encapsulated liposomes has been demonstrated to improve energy state and function of the cold-stored liver [6,7,13].
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耗材:滤纸、PVDF膜或NC膜、乳胶手套等
仪器:电泳仪、电转槽、摇床、制冰机(电转时需冰浴)等
原理简介:
本试剂盒采用改进SDS-碱裂解法裂解细胞,离心吸附柱内的硅基质膜在高盐,低pH值状态下选择性地结合溶液中的质粒DNA,再通过去蛋白液和漂洗液将杂质和其它细菌成分去除,最后低盐,高pH值的洗脱缓冲液将纯净质粒DNA从硅基质膜上洗脱。
注意事项:
◆ 第一次使用时,将试剂盒所带全部的RNase A加入溶液P1后(终浓度100ug/ml)置于4℃保存。如果溶液P1中RNase A失活,提取的质粒可能会有混杂有微量RNA残留, 这时可在溶液P1中补加RNase A即可。
◆ 第一次使用前请先在15ml漂洗液WB中加入45ml无水乙醇,加入后请及时在方框打钩标记已加入乙醇,以免多次加入!
◆ 温度低时溶液P2中SDS可能会出现浑浊或者析出沉淀,可在37℃水浴加热几分钟,即可恢复澄清,不要剧烈摇晃,以免形成过量的泡沫。
◆ 避免试剂长时间暴露于空气中产生挥发、氧化、pH值变化,各溶液使用后应及时盖紧盖子。
试剂盒特点:
◆ 产量高---一次提取高达30ug以上的质粒。
◆ 纯度高---OD260/OD280一般为1.80~1.85本试剂盒提取的质粒纯度好,能充分保证测序所需要的读长(用于ABI3730测序一般可达1000bp有效读长)。
◆ 快速,方便,不需要使用有毒的苯酚,氯仿等试剂,也不需要乙醇沉淀。
提示
BIOTEKE的质粒提取试剂盒既适用于革兰氏阴性菌中质粒的提取,同时也可从革兰氏阳性菌中提取质粒。由于革兰氏阳性菌外被一层较厚的细胞壁,会严重阻碍细菌细胞的裂解,因此必须在裂解细胞前破除,方法如下:
收集适量的菌体,加入250ul溶液P2,充分悬浮菌液,加入溶菌酶使其终浓度在10-20mg/ml左右在37℃处理30分钟左右。加入溶菌酶的浓度和处理的时间可根据不同的菌主和具体实验条件进行调整。
原液分装。抗体孵育之前稀释。
做WB的浓度不一定需要很高的。可以设置简单梯度1:200;1:500;1:1000 。看结果再优化
1. 使用预染 Marker,不过分子量不是特别准;
2. 所使用的 Marker 条带与待测蛋白质带有相同的抗原表位,比如都带有 His 融合标签;
3. 可以使用普通的 Marker 转膜使用 丽春红 等染色,然后在膜上标记出 Marker 各条带的位置。

