EdU Imaging Kits (DAB)
mRNA synthesis
In vitro transcription of capped mRNA with modified nucleotides and Poly(A) tail
Tyramide Signal Amplification (TSA)
TSA (Tyramide Signal Amplification), used for signal amplification of ISH, IHC and IC etc.
Phos Binding Reagent Acrylamide
Separation of phosphorylated and non-phosphorylated proteins without phospho-specific antibody
Cell Counting Kit-8 (CCK-8)
A convenient and sensitive way for cell proliferation assay and cytotoxicity assay
SYBR Safe DNA Gel Stain
Safe and sensitive stain for visualization of DNA or RNA in agarose or acrylamide gels.
Inhibitor Cocktails
Protect the integrity of proteins from multiple proteases and phosphatases for different applications.
测量细胞增殖和细胞周期是评估细胞健康、确定遗传毒性和评估药物药效的基本方法,常用的方法是直接测量DNA 的合成。在以往的实验中常用方法包括掺入放射性核苷(3H-thymidine)或BrdU等。而EdU Imaging Kits (DAB)则采用了一种新的方法:点击化学-CuAAC(铜催化的叠氮化物-炔烃环加成反应),使用该反应可直接测量在细胞周期S期的DNA合成。
在基于BrdU的检测中,需要额外的DNA变性(通常使用HCl、加热或用DNase消化)以使BrdU暴露于抗BrdU抗体。胸腺嘧啶核苷类似物EdU(5-ethynyl-2’-deoxyuridine)是BrdU的理想替代品,因为它不受苛刻的DNA变性条件的影响,EdU可以在DNA合成过程中掺入DNA链中。EdU的炔基是一种生物惰性基团,可以通过CuAAC反应与染料的叠氮基发生极强的选择性反应,从而生成1, 2, 3-三唑产物。EdU和Biotin azide具有生物学上独特的基团,其连接后可以标记增殖细胞的DNA,具有低背景和高检测灵敏度。这种CuAAC反应可在极温和的条件下提供出色的区域选择性和定量转化。
EdU Imaging Kits (DAB) 采用的是biotin azide与EdU进行连接后特异性标记增殖细胞的DNA,然后加入辣根过氧化酶标记的链霉亲和素(HRP-Streptavidin)和biotin结合,通过 DAB显色,最后可通过显微镜进行观察。
图1:本试剂盒检测原理图
图2:本试剂盒检测验证图
无EdU组观察不到细胞染色,使用EdU后明显观察到细胞的棕色染色,而这种染色被加入的Hydroxyurea(DNA合成抑制剂)所抑制
Components | 50-500 T | 200-2000 T |
EdU 10mM (Component A) | 200 μL | 800 μL |
Biotin azide (Component B) | 55 μL | 220 μL |
10X EdU Reaction Buffer (Component C) | 3.5 mL | 13 mL |
CuSO4 (100 mM Aqueous Solution) (Component D) | 1.2 mL | 4.8 mL |
10X EdU Buffer Additive (Component E) | 1 vial | 4 vials |
Streptavidin-HRP (Component F) | 220 μL | 900 μL |
Streptavidin-HRP Diluent (Component G) | 10 mL | 40 mL |
DAB Chromogen Solution A | 10 mL | 40 mL |
DAB Chromogen Solution B | 10 mL | 40 mL |
Store the kit at -20ºC away from moisture, stable for 1 year. Biotin azide and DAB Chromogen Solutions should be stored away from light. |