
Bromoenol lactoneInhibitor of myocardial cytosolic calcium-independent phospholipase A2 (iPLA2) |
Sample solution is provided at 25 µL, 10mM.
































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Chemical structure


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Cas No. | 88070-98-8 | SDF | Download SDF |
Synonyms | N/A | ||
Chemical Name | 6E-(bromomethylene)tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one | ||
Canonical SMILES | Br/C=C1CCC(C(=O)O1)c1cccc2ccccc12 | ||
Formula | C16H13BrO2 | M.Wt | 317.2 |
Solubility | ≤2mg/ml in ethanol;25mg/ml in DMSO;50mg/ml in dimethyl formamide | Storage | Store at -20°C |
Physical Appearance | A crystalline solid | Shipping Condition | Evaluation sample solution : ship with blue ice.All other available size:ship with RT , or blue ice upon request |
General tips | For obtaining a higher solubility , please warm the tube at 37 ℃ and shake it in the ultrasonic bath for a while.Stock solution can be stored below -20℃ for several months. |
Bromoenol lactone is a potent and irreversible inhibitor of myocardial cytosolic calcium-independent phospholipase A2 (iPLA2) [1].
The iPLA2 has been involved in stimulus-induced arachidonic acid release and lysophospholipid generation. The catalytic action of iPLA2 is responsible for phospholipid remodeling as a housekeeping function. Arachidonic acid and lysophospholipid generated by iPLA2 act as a signaling molecule in cellular functions, including eicosanoid production, glucose-induced insulin secretion, Fas-induced apoptosis, cellular proliferation, membrane traffic in fusion, contribution to myocardial ischemia, and others [2].
BEL promoted apoptosis in a variety of cell lines, including U937, THP-1, and MonoMac (human phagocyte), RAW264.7 (murine macrophage), Jurkat (human T lymphocyte), and GH3 (human pituitary). Long term treatment with BEL (up to 24 h) increased annexin-V binding to the cell surface and nuclear DNA damage. BEL induced the proteolysis of procaspase-9 and procaspase-3 and increased cleavage of poly (ADP-ribose) polymerase [1]. BEL inhibited cellular phosphatidic acid phosphohydrolase (PAP) activity in intact P388D1 macrophages with an IC50 of ~8 μM. BEL blocked triacylglycerol biosynthesis in P388D1 cells by decreasing diacylglycerol availability [3].
References:[1] Fuentes L, Pérez R, Nieto M L, et al. Bromoenol lactone promotes cell death by a mechanism involving phosphatidate phosphohydrolase-1 rather than calcium-independent phospholipase A2[J]. Journal of Biological Chemistry, 2003, 278(45): 44683-44690.[2] Akiba S, Sato T. Cellular function of calcium-independent phospholipase A2[J]. Biological and Pharmaceutical Bulletin, 2004, 27(8): 1174-1178.[3] Balsinde J, Dennis E A. Bromoenol lactone inhibits magnesium-dependent phosphatidate phosphohydrolase and blocks triacylglycerol biosynthesis in mouse P388D1 macrophages[J]. Journal of Biological Chemistry, 1996, 271(50): 31937-31941.
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逆转录(reverse transcription)是以RNA为模板合成DNA的过程,即RNA指导下的DNA合成。是RNA病毒的复制形式,需逆转录酶的催化。艾滋病病毒(HIV)就是一种典型的逆转录病毒。
逆转录与反转录严格意义上来说没有什么区别,但是逆转录是RNA类病毒自主行为,在整合到宿主细胞内以RNA为模板形成DNA的过程;反转录是进行基因工程过程中,人为地提取出所需要的目的基因的信使RNA,并以之为模板人工合成DNA的过程。二者虽同为RNA→DNA的过程,但地点不同,相对性的来说,逆转录在体内,反转录在体外。
2、取灭过菌且无核酸酶的0.2ml离心管,依次加入2~5μgRNAnμL
3、65℃保温5min,然后冰浴5min;
4、往3步骤中的0.2ml离心管依次加入下列组份
RNase抑制剂(40u/μL)0.5μL
10×M-MLVReactionBuffer2μL
DTT(200mM)1μL
逆转录酶(M-MLV)1μL
5、轻轻混匀后,然后2000rpm离心20s;
6、先在37℃保温1hr,然后70℃保温15min;
7、上述产物可立即进行下一步的PCR反应或-20℃保存。向左转|向右转
在进行RT反应之前,应考虑以下几个方面:
1、RNA
成功的cDNA合成来自高质量的RNA,高质量的RNA至少应保证全长并且不含逆转录酶的抑制剂,如EDTA或SDS。在提取RNA的过程中,要特别防止RNase的污染,同时在逆转录反应中经常加入RNase抑制剂以增加cDNA合成的长度和产量。RNase抑制剂要在第一链cDNA合成反应中,在缓冲液和还原剂(如DTT)存在的条件下加入,因为cDNA合成前的过程会使抑制剂变性,从而释放结合的可以降解RNA的RNase。蛋白RNase抑制剂仅防止RNaseA,B,C对RNA的降解,并不能防止皮肤上的RNase,因此尽管使用了这些抑制剂,也要小心不要从手指上引入RNase,实验过程中经常更换新手套。
2、引物的选择
OligodT
选择OligodT时,要求RNA必须有PolyA,所以真核生物的mRNA都适用。适合长链甚至全长mRNA的RT,所以对RNA样品的质量要求较高,最好不要有明显的DNA污染、RNA降解和RNA断裂。假如想探索新的mRNA进行RT反应,建议推荐使用OligodT引物。使用OligodT引物要比随机引物和特异性引物的稳定性要好。
随机引物
适合各种RNA的RT,尤其适合模板丰度很低的情况(比如某个gene表达量很低)。选择随机引物时,第一链cDNA合成反应中就是以所有的RNA为模板,然后进行PCR反应时设计引物进行特异性扩增。同时要注意随机引物的量和总RNA量之间的关系,一般建议每5μg总RNA的随机引物的用量为50ng,如果每5μg总RNA的随机引物的用量超过250ng,可能会导致小片段产物(<500bp)的增加和长片断、全长产物产物的降低。
特异性引物
特异性引物只能用你设计引物时的下游引物做RT,引物设计质量影响RT的结果,而且不同引物退火温度本来就不相同,所以按照说明书按照一个温度做不是最佳选择,一般不推荐。向左转|向右转

