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Isolation, Purification and Identification of the Human DNA Polymerase γ

Author: ZhangYingHui
Tutor: LinJuSheng
School: Huazhong University of Science and Technology
Course: Internal Medicine
Keywords: Human cervical carcinoma cell DNA polymerase γ Mitochondria Ion exchange chromatography Purification
CLC: R341
Type: Master's thesis
Year: 2008
Downloads: 32
Quote: 0
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Abstract


Objective: Mitochondrial DNA was extracted from human cervical cancer cells (Hela) polymerase γ (mitochondrial DNA polymerase, Polγ), purified and identified by the purity and activity for mitochondrial toxicity in vitro drug . Methods : the use of ion exchange chromatography method to extract purified Hela cell mitochondria mitochondrial DNA polymerase gamma , and with Bradford method detecting protein concentration , the SDS-PAGE analysis of protein purity and the relative molecular weight , Western-Blot authentication protein with α-32P- dTTP incorporation , a liquid scintillation counter for radioactivity measurements to determine the activity of DNA polymerase γ . Results: We successfully extracted and purified DNA polymerase gamma Hela cells , an approximately 140KD Subunit identified by SDS-PAGE , Western-Blot confirmed enzyme . After a series of chromatography , polymerase γ available 150 -fold purification , the yield of 6% , the total activity of the enzyme 4.81ukat specific activity 36.17ukat/mg . Conclusion : Hela cells Polγ by ion exchange chromatography, after extraction and purification of higher activity can be used for in vitro drug mitochondrial toxicity testing .

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CLC: > Medicine, health > Basic Medical > Human biochemistry, molecular biology
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