Page 10 - 2016 July 專刊-H-0722

【 Anti-Poly ADP-Ribose Polymerase 】 ROC-11835238001
偵測DNA 斷裂

偵測原理與標的  實驗的操作流程
利用 Anti-PARP的抗體來辨識PARP被蛋白
偵測
酶水解後的蛋白片段 (85KDa)。PARP本是
一個分子量116KDa的蛋白質，細胞凋亡早 細胞凋亡的晚期會造成DNA的斷裂，研究發現細胞
期，會專一性的結合到DNA斷裂的位置， 凋亡時，會活化細胞的核酸內切酶，對特定的核酸
PRAP 被水解後會產生 85KDa與24KDa兩個 進行切割，將DNA切為180bp 左右的小片段。因此
活化 片段；因此偵測85KDa的片段可以當作細胞 偵測小片段的核酸 (LMWDNA) 也就發展為細胞凋
Caspase
凋亡的早期指標。 亡晚期的鑑別指標。過程為了偵測DNA的斷裂現象，
大多數的方法會採去將細胞內的核酸“預先標記”

適用的偵測檢體 適用的偵測方法
--Cell lysates --Western blotting 利用 [3H]-thymidine 放射性物質或 BrdU 預先標誌核酸；在 DNA 複製時；這些預先標誌的核酸會被帶
入 genomic DNA，再給予 Apoptosis 誘導物刺激後；這些被標誌的核酸將會斷裂而被偵測到。研究 Cell
mediated Cytotoxicity，細胞內的核酸片段會在給予細胞刺激前先被標誌，當細胞受到毒殺作用時，細
The appearance of a large (89 kD) cleavage fragment is indicative of caspase proteolytic activity. 胞膜會破損，因此小片段的核酸(LMW)會由細胞質釋放到Supernatant。

Detection of cleaved PARP in cell extracts of apoptotic CEM T cells.
CEM T cells were incubated with one of three apoptosis-inducing
drugs. Cell extracts from 3 x 105 treated or untreated cells were Distribution of HMW and LMW DNA in cells undergoing apoptosis and target cells during cell
fractionated on an 10% polyacrylamide gel in the presence of SDS. mediated cytotoxicity.
After electrophoresis, proteins on the gel were transferred to a
PVDF membrane by electroblotting and the blot was blocked with A p o o t p s i s C l l e m e e t a i d c d o t o t y y t i c i x
5% powdered milk. The blocked membrane was incubated with a
1:3000 dilution of Anti-PARP. Subsequent incubations with a Compartment HMW DNA LMW DNA HMW DNA LMW DNA
peroxidase-conjugated anti-rabbit secondary antibody and a
peroxidase substrate revealed the presence of PARP cleavage Nucleus + + + +
products on the blot. Note that the antibody recognizes both
uncleaved PARP (113 kD) and the larger cleavage fragment (89 Cytoplasm - + - +
kD).
Lane 1: Untreated control cells Supernatant - - - +
Lane 2: Cells treated with 100 ng/ml doxorubicin for 24 h
Lane 3: Cells treated with 1 mg/ml methotrexate for 24 h
Lane 4: Cells treated with 1 mg/ml cytarabin for 24 h. Compartmentalization of HMW and LMW DNA in normal and apoptotic cells.

Normal cell Apoptotic cell
References HMW LMW HMW LMW
1. A Functional Nuclear Localization Signal in Insulin-Like Growth Factor Binding Protein-6 Mediates Its Nuclear Import
Cristiana Iosef, Theofanis Gkourasas, Christina Y. H. Jia, Shawn S.-C. Li, and Victor K. M. Han Endocrinology, Mar 2008; 149: 細胞核 + - 減少 增加
1214 - 1226.
2. Acetyl-Keto-b-Boswellic Acid Induces Apoptosis through a Death Receptor 5–Mediated Pathway in Prostate Cancer Cells 細胞質 - - - 增加
Min Lu, Lijuan Xia, Huiming Hua, and Yongkui Jing Cancer Res., Feb 2008; 68: 1180 - 1186 Normal cell Apoptotic cell

利用細胞代謝方法偵測斷裂 DNA；只適用於可以在體外增生的細胞株，例如：Cell lines。有些細胞無
【Caspase 相關抗體】 法在體外進行 DNA 複製，無法採用這個方法。因此發展出直接從細胞分離出小片段 DNA；再利用電
泳的方式來偵測 LMW-DNA 的方式，EX: Apoptotic DNA Ladder Kit
T g r a t e S p e e i c s A p c i l p o i t a n C N . t a . o P a c k a g e
Hu, Rat ,Rb, Pig, 偵 測 方 法 相 關 產 品 產 品 編 號
Anti-Caspase 3, active WB,IF SI-C8487 200ul
Dog, Bovine 膠體電泳 A p o D c i t o t p N L A a d d t i K r e R O C 1 - 1 8 3 5 2 4 6 0 0 1
Anti-Caspase 3, clone84803 Hu WB SI-C5737 100ug E S I L A C D l l e e D h t a e t e o i t c n E S I L P A L U S R O C 1 - 1 7 7 4 4 2 5 0 0 1

Anti-Caspase 6 Hu WB,ARP SI-C7599 200ul In Situ CellDeath Detection Kit, Fluorescein (TUNEL) ROC-11684795910
Anti-Caspase 8 (pro) Hu WB,ARP SI-C7849 200ul 螢光顯微鏡 In Situ Cell Death Detection Kit, TMR red (TUNEL) ROC-12156792910

Anti-Caspase 8 Hu WB, E, ARP SI-C4106 200ul In Situ Cell Death Detection Kit, AP (TUNEL) ROC-11684809910
可見光顯微鏡
Anti-Caspase 9 Hu,Rat WB,IHC,IP,ARP SI-C7729 200ul In Situ Cell Death Detection Kit, POD (TUNEL) ROC-11684817910

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