Enzymatic Activity Assays in Yeast Cell Extracts
酵母细胞提取物的酶活性分析
发布: 2014年12月05日第4卷第23期 DOI: 10.21769/BioProtoc.1312 浏览次数: 11815
评审: Fanglian He
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参见作者原研究论文
The authors used this protocol in:
May 2014
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Abstract
Saccharomyces cerevisiae (S. cerevisiae) (commonly known as baker’s yeast) is a model organism that has a similar upstream base excision repair (BER) pathway for the repair of methylated bases as that in mammalian cells, and it is very easy to maintain in the laboratory environment. Here, we described a method to prepare cell extracts from yeast to investigate their enzymatic activities. This protocol is a quick and efficient way to make yeast cell extracts without using commercial kits.
Keywords: DNA damage (DNA损伤)
DNA polymerase (DNA聚合酶)
DNA repair (DNA修复)
Base excision repair (碱基切除修复)
Ligation failure (结扎失败)
Materials and Reagents
- EDTA-free protease inhibitor cocktail tablet (Roche Diagnostics, catalog number: 11836170001 )
- Bio-Rad protein assay dye reagent concentrate (Bio-Rad Laboratories, catalog number: 500-0006 )
- Dextrose (Sigma-Aldrich, catalog number: D9434 )
- Bacto-peptone (Sigma-Aldrich, catalog number: P5905 )
- Bacto-yeast extract (Sigma-Aldrich, catalog number: Y1625 )
- Adenine sulfate (Sigma-Aldrich, catalog number: A3159 )
- Trizma base (Sigma-Aldrich, catalog number: T1503 )
- EDTA (Sigma-Aldrich, catalog number: 93283 )
- NaCl (Sigma-Aldrich, catalog number: S7653 )
- β-mercaptoethanol (Sigma-Aldrich, catalog number: B0126 )
- Formamide (Sigma-Aldrich, catalog number: F9037 )
- Bromophenol blue (Sigma-Aldrich, catalog number: B0126)
- Xylene cyanol (Sigma-Aldrich, catalog number: X4126 )
- Dithiothreitol-DTT (Sigma-Aldrich, catalog number: D0632 )
- Sodium borohydride-NaBH4 (Sigma-Aldrich, catalog number: 247677 )
- Sterile water
- Yeast peptone dextrose adenine (YPDA) medium (see Recipes)
- Potassium phosphate buffer (see Recipes)
- Lysis buffer (see Recipes)
- 10x reaction buffer (see Recipes)
- Gel loading buffer (see Recipes)
Equipment
- 0.5 mm diameter glass beads (Bio Spec Products, catalog number: 11079105 )
- Eppendorf tubes
- Table-top centrifuge
- Table-top heat block
Procedure
文章信息
版权信息
© 2014 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Çağlayan, M. and Wilson, S. H. (2014). Enzymatic Activity Assays in Yeast Cell Extracts. Bio-protocol 4(23): e1312. DOI: 10.21769/BioProtoc.1312.
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分类
微生物学 > 微生物生物化学 > 蛋白质 > 活性
生物化学 > 蛋白质 > 活性
分子生物学 > DNA > DNA 损伤和修复
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