The fragmentation of genomic DNA by cellular nucleases during the later stages of apoptosis is also one of the most easily measured features of apoptotic cells. Nuclease activity generates DNA fragments ranging from ~300 bp to 50 bp in length, resulting in a typical DNA "˜laddering"™ appearance when analyzed by agarose gel electrophoresis. These fragments have exposed 3"™-hydroxyl (OH) ends which can be labeled with deoxyuridine triphosphates (dUTP). An enzyme, terminal deoxynucleotidyl transferase (TdT), is used to catalyze the template-independent addition of fluorescent tagged dUTP to the 3"™-OH ends of double or single stranded DNA. This method is often called TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) or end labeling.
With the APO-DIRECTTM Kit, cells are stained with FITC-labeled dUTP in a single step. Samples can then be analyzed via flow cytometry. Samples that are apoptotic will stain brightly due to the substantial number of exposed 3"™-OH sites, while cells that are non-apoptotic will not have incorporated significant amounts of FITC-dUTP and will stain dimly.
The APO-DIRECT Kit is shipped in one container and consists of two packages. Upon arrival one should be stored at 2-8C and the other at -20C.
Li X, Traganos F, Melamed MR and Darzynkiewicz Z. 1995. Cytometry. 20(2): 172-180.
Tomei LD and Cope FD, eds. 1991. Current Communications in Cell and Molecular Biology. 3: 47-60. Cold Springs Harbor, NY.
Arends MJ, Morris RG and Wyllie AH. 1990. Am J Pathol. 136(3): 593-608.
Darzynkiewicz Z, Crissman HA and Robinson JR, eds. 1994. Methods in Cell Biology: Flow Cytometry Second Edition. 41: 15-38. Academic Press Inc.
Eschenfeldt WH, Puskas RS and Berger SL. 1987. Methods Enzymol. 152: 337-342.
Darzynkiewicz Z, Bruno S, Del Bino G, Gorczyca W, Hotz MA, Lassota P and Traganos F. 1992. Cytometry. 13(8): 795-808.
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其检测原理为：在正常的活细胞中，磷脂酰丝氨酸（phosphotidylserine，PS）位于细胞膜的内侧，但在早期凋亡的细胞中，PS 从细胞膜的内侧翻转到细胞膜的表面，暴露在细胞外环境中。Annexin-Ⅴ（膜联蛋白-V）是一种分子量为35-36KD的Ca2+ 依赖性磷脂结合蛋白，能与PS高亲和力结合。可通过细胞外侧暴露的磷脂酰丝氨酸与凋亡早期细胞的胞膜结合。
操作步骤：
1.从室温平衡20min后的铝箔袋中取出所需板条，剩余板条用自封袋密封放回4℃。
2.设置标准品孔和样本孔，标准品孔各加不同浓度的标准品50μL；
3.样本孔中加入待测样本50μL；空白孔不加。
4.除空白孔外，标准品孔和样本孔中每孔加入辣根过氧化物酶（HRP）标记的检测抗体100μL，用封板膜封住反应孔，37℃水浴锅或恒温箱温育60min。
5.弃去液体，吸水纸上拍干，每孔加满洗涤液（350μL），静置1min，甩去洗涤液，吸水纸上拍干，如此重复洗板5次（也可用洗板机洗板）。
6.每孔加入底物A、B各50μL，37℃避光孵育15min。
7.每孔加入终止液50μL，15min内，在450nm波长处测定各孔的OD值。
不知道发在这里合适不，实在是求助无门啊！版主手下留情。
想请问下那个公司有专门的蛋白质荧光标记试剂盒出售。最好的是CY5的，我准备做三标。性价比越高越好
那些做过的前辈们指导一下。
2、 血浆：EDTA、柠檬酸盐、肝素血浆可用于检测。1000×g离心30分钟去除颗粒。
3、 细胞上清液：1000×g离心10分钟去除颗粒和聚合物。
4、 组织匀浆：将组织加入适量生理盐水捣碎。1000×g离心10分钟，取上清液。
5、 保存：如果样品不立即使用，应将其分成小部分-70℃保存，避免反复冷冻。尽可能的不要使用溶血或高血脂血。如果血清中大量颗粒，检测前先离心或过滤。不要在37℃或更高的温度加热解冻。应在室温下解冻并确保样品均匀地充分解冻。
此IBL试剂盒能用于小鼠血清,EDTA血浆,细胞上清中白介素-6的定量检测 试剂盒成分 1 预包被板: 抗小鼠白介素-6兔子IgG,亲合纯化 96T 2 酶标记抗体: (30倍浓缩)HRP标记抗小鼠白介素-6兔子IgG,亲合纯化 0.4mL x 1 3 标准品: 重组小鼠白介素-6 0.5mL x 2 4 EIA缓冲液: 含1% BSA, 0.05%吐温20 BPS 30mL x 1 5 标记抗体稀释液: 含1% BSA, 0.05%吐温20 BPS 12mL x 1 6 显色剂: TMB底物液 15mL x 1 7 终止液: 1N硫酸 12mL x 1 8 浓缩洗涤液: (40倍浓缩) 含1% BSA, 0.05%吐温20 BPS 50mL x 1 操作说明 1实验所需器材(但试剂盒没有提供) 酶标仪(450nm) 微移液管及其吸嘴 量筒及烧杯 去离子水 冰箱(4°C) 坐标纸(log/log) 吸水纸 试管(用于标准品稀释) 温育箱(37°C ± 1°C) 洗瓶 (用于洗板) 一次性试剂管(用于浓缩酶标记抗体和显色剂)向左转|向右转
