Staining Procedure for Flow Cytometric Detection of Human Cyclins | |
This is a standard protocol used at Pharmingen for Quality Control testing of the anti-cyclin antibodies by flow cytometry. It is a good starting point. However, investigators may need to optimize protocols for their own experimental system. It is particularly useful to refer to the published literature regarding protocols typically used for a given type of protein. MATERIALS: BUFFERS: REAGENTS: Wash buffer stored at 4°C, 75% ethanol stored at –20°C, Pure methanol stored at -20°C, 1% formaldehyde (methanol-free) in PBS, stored at 4°C, 0.25% Triton ® X-100 in Wash buffer, stored at 4°C, propidium iodide (PI) solution: 10 µg/ml PI in PBS, stored at 4°C. Procedure: I.Fixation 1.Harvest, count and pellet cells following standard procedures.2.Wash cells once by resuspending the pellet in 30-40 ml of Wash buffer. Centrifuge at 200 x g for 10 min and aspirate supernatant.3.While vortexing, add 10 ml cold 75% ethanol (for D1, use pure cold methanol), drop by drop, to the cell pellet.4.Incubate at –20°C for a minimum of 2 hr. The fixed cells can be stored at –20°C in 75% ethanol for up to 30 days.Note: For D-type cyclins (besides D1) the cells should first be fixed in 5 ml of 1% methanol-free formaldehyde in PBS for 15 min on ice (or at 4°C) prior to fixation with ethanol. Following incubation with formaldehyde, centrifuge at 200 x g for 10 min and aspirate supernatant. Resuspend pellet in 30-40 ml Wash Buffer, centrifuge at 200 x g for 10 min and aspirate supernatant. Then go to Step No. 3 above.II.Staining 1.Just prior to staining, remove ethanol by centrifugation at 200 x g for 10 min. Aspirate and wash once by resuspending pellet in 30-40 ml of Wash buffer. Centrifuge at 200 x g for 10 min. Aspirate supernatant.2.Add 5 ml cold 0.25% Triton ® X-100 in Wash buffer to the cell pellet, vortex and incubate 5 min on ice (or at 4°C).3.Add 30-40 ml Wash buffer to the above suspension and centrifuge at 200 x g for 10 min. Aspirate supernatant.4.Resuspend pellet in Wash buffer to a final concentration of 1 x 107 cells/ml.5.Aliquot 100 ml cell suspension (1 x 106 cells) into 12 x 75 mm tubes for staining.6.Add 20 ml of anti-cyclin or isotype control antibody at optimal working dilution. Incubate 30 min at RT in the dark.7.Add 2 ml Wash buffer, centrifuge for 5 min at 200 x g. Aspirate supernatant.8.If using directly conjugated isotype controls and mAbs, go to Step 10 below.9.If using unconjugated isotype controls and mAbs, add 20 ml of FITC-conjugated goat anti-mouse Ig (Cat. No. 12064D) at optimal working dilution. Incubate 30 min at RT in the dark. Add 2 ml Wash buffer, centrifuge 5 min at 200 x g Aspirate supernatant.10.Resuspend cell pellet in 0.5 ml PI solution for simultaneous analysis of DNA cell cycle and cyclin expression. Incubate cells at 4°C in PI for 20 min prior to analyzing by flow cytometry. Analyze stained cells within 4 hr. Store at 4°C in the dark prior to analysis. |
蛋白质作为构成人体组织器官的支架和主要物质，在人体生命活动中起着重要作用。蛋白质的相互作用能产生许多效应，如形成特异底物作用通道、生成新的结合位点、失活、作用底物专一性和动力学变化等，细胞的代谢、信号......
2021年9月9日，无锡臻和生物科技有限公司（以下简称“臻和科技”）与美国VyantBio公司签署TissueofOrigin®（以下简称“TOO®”）全球权益和ZL转让协议，全资收购这款唯一获FDA......
2021年7月20日，JournalofCellularPhysiology及JournalofCellularBiochemistry同时撤回了中国学者49篇文章。从2019年开始，Journalo......
近日，昆士兰科技大学（QUT）领导的一项研究强调了衣原体感染对男性生育能力的潜在影响。该研究首次在不育男性睾丸组织活检中发现了衣原体的存在。研究人员还在18位志愿者的血液中发现了12种针对沙眼衣原体细......
磷酸甘油酸突变酶1（PGAM1）通过其代谢活性以及与其他蛋白质（例如α平滑肌肌动蛋白（ACTA2））的相互作用，在癌症代谢和肿瘤进展中起关键作用。变构调节被认为是发现针对PGAM1的高选择性和有效抑制......
分析测试百科网讯，今年来化工厂的频繁爆炸事件造成极大社会反响，化工生产中的安全问题再次触动了所有国人的神经。安全排查、人员责任等手段固然能起到一定的效果，但还不能从根本上解决问题；分析诸多事件，我们发......
2018年12月6日，来自圣迭戈的消息——Illumina公司（纳斯达克股票代码：ILMN）今天宣布推出新型高密度基因分型芯片Infinium™GlobalDiversityArray。这款芯片设计源......
当地时间11月30日，科学狂人文特尔（J.CraigVenter）创办的世界上最大的DNA测序实验室HumanLongevity公司宣布收购生物标志物开发企业CypherGenomics，具体的财务细......
这里描述了按照USEPA方法OIA-16771-4，通过价位交换、流动注射分析(FIA)和安培计检测有效氰化物的方法。这个方法用于USEPA关于清洁水法案，资源保护与回收法案，全面环境响应，补偿与责任......
SDS-PAGE异常电泳现象及分析SDS-PAGEHallofShame.pdf 很不错的东东~~推荐下~......