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. |
蛋白質作為構成人體組織器官的支架和主要物質，在人體生命活動中起著重要作用。蛋白質的相互作用能產生許多效應，如形成特異底物作用通道、生成新的結合位點、失活、作用底物專一性和動力學變化等，細胞的代謝、信號......
2021年9月9日，無錫臻和生物科技有限公司（以下簡稱“臻和科技”）與美國VyantBio公司簽署TissueofOrigin?（以下簡稱“TOO?”）全球權益和ZL轉讓協議，全資收購這款唯一獲FDA......
2021年7月20日，JournalofCellularPhysiology及JournalofCellularBiochemistry同時撤回了中國學者49篇文章。從2019年開始，Journalo......
近日，昆士蘭科技大學（QUT）領導的一項研究強調了衣原體感染對男性生育能力的潛在影響。該研究首次在不育男性睪丸組織活檢中發現了衣原體的存在。研究人員還在18位志愿者的血液中發現了12種針對沙眼衣原體細......
磷酸甘油酸突變酶1（PGAM1）通過其代謝活性以及與其他蛋白質（例如α平滑肌肌動蛋白（ACTA2））的相互作用，在癌癥代謝和腫瘤進展中起關鍵作用。變構調節被認為是發現針對PGAM1的高選擇性和有效抑制......
分析測試百科網訊，今年來化工廠的頻繁爆炸事件造成極大社會反響，化工生產中的安全問題再次觸動了所有國人的神經。安全排查、人員責任等手段固然能起到一定的效果，但還不能從根本上解決問題；分析諸多事件，我們發......
2018年12月6日，來自圣迭戈的消息——Illumina公司（納斯達克股票代碼：ILMN）今天宣布推出新型高密度基因分型芯片Infinium?GlobalDiversityArray。這款芯片設計源......
當地時間11月30日，科學狂人文特爾（J.CraigVenter）創辦的世界上最大的DNA測序實驗室HumanLongevity公司宣布收購生物標志物開發企業CypherGenomics，具體的財務細......
這里描述了按照USEPA方法OIA-16771-4，通過價位交換、流動注射分析(FIA)和安培計檢測有效氰化物的方法。這個方法用于USEPA關于清潔水法案，資源保護與回收法案，全面環境響應，補償與責任......
SDS-PAGE異常電泳現象及分析SDS-PAGEHallofShame.pdf 很不錯的東東~~推薦下~......