NP-40 Fractionation and Nucleic Acid Extraction in Mammalian Cells
NP-40 提取分选哺乳动物细胞核酸
发布: 2017年10月20日第7卷第20期 DOI: 10.21769/BioProtoc.2584 浏览次数: 14276
评审: Gal HaimovichAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Jun 2014
Abstract
This technique allows for efficient, highly purified cytoplasmic and nuclear-associated compartment fractionation utilizing NP-40 detergent in mammalian cells. The nuclear membrane is not disturbed during the fractionation thus leaving all nuclear and perinuclear associated components in the nuclear fraction. This protocol has been modified from Sambrook and Russell (2001) in order to downscale the amount of cells needed. To determine the efficiency of fractionation, we recommend using qPCR to compare the subcellular compartments that have been purified with equivalent amount of control whole cell extracts.
Keywords: Cell fractionation (细胞分级分离)
Cytoplasmic and nuclear extractions (细胞质和细胞核提取)
Nucleic acid purification (核酸纯化)
Quantitative PCR (定量PCR)
Background
To fully obtain an understanding of cellular processes, fractionation of nuclear and cytoplasmic compartments are needed. There are many protocols and even some commercial kits available to help separate the two compartments. However, most require high centrifugation speeds and there is a high discrepancy in the yield and even the methods to verify the amount of contamination in the final products. Our protocol utilizes a small benchtop centrifuge at low speeds to obtain highly pure extractions for the cytoplasmic and a combined nuclear/perinuclear associated compartments as well as the data analysis to verify the percentage of contamination. To date, the cells lines that have been tested are 293 T, HeLa and GHOST cell lines. (Galvis, 2014; Galvis et al., 2014).
Materials and Reagents
- BD 21 G needles (Fisher Scientific, catalog number: 14-823-55)
Manufacturer: BD, catalog number: 305274 . - BD tuberculin syringes (Fisher Scientific, catalog number: 14-823-2F)
Manufacturer: BD, catalog number: 309602 . This product has been discontinued. - TipOne 10 µl pipet tips (USA Scientific, catalog number: 1111-3200 )
- TipOne 1-200 µl natural pipet tips (USA Scientific, catalog number: 1111-1800 )
- TipOne 1,000 µl natural pipet tips (USA Scientific, catalog number: 1111-2020 )
- Costar 6-well cell culture plates (Fisher Scientific, catalog number: 07-200-80)
Manufacturer: Corning, catalog number: 3506 . - 1.5 ml microcentrifuge tubes (Fisher Scientific, catalog number: 14-666-318 )
- Iscove’s modification of DMEM (Mediatech, catalog number: 10-016-CV )
- Tween 40 (CHEM-IMPEX INTERNATIONAL, catalog number: 01513 )
- 1x phosphate buffered saline (PBS) (MP Biomedicals, catalog number: 091860454 )
- 1x trypsin-EDTA (Mediatech, catalog number: 25-051-Cl )
- 0.5 M ethylenediaminetetraacetic acid (EDTA) pH 8.0 (Fisher Scientific, catalog number: BP2482-500 )
- 10% sodium dodecyl sulfate (SDS) (Mediatech, catalog number: 46-040-Cl )
- Proteinase K (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: EO0491 )
- Ribonuclease A, DNase free (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: EN0531 )
- Potassium acetate (Fisher Scientific, catalog number: P171-500 )
- Isopropanol (Fisher Scientific, catalog number: A416-500 )
- Ethanol 70% (Fisher Scientific, catalog number: BP82011 )
- Sodium hydroxide (NaOH) (Fisher Scientific, catalog: S318-500 )
- SYBR Green PCR Master mix (Thermo Fisher Scientific, Applied BiosystemsTM, catalog number: 4309155 )
- Magnesium chloride (MgCl2) (Fisher Scientific, catalog number: BP214-500 )
- Sucrose (Fisher Scientific, catalog number: S3-500 )
- Tris-HCl buffer pH 7.4 (Lonza, catalog number: 51237 )
- Potassium chloride (KCl) (Fisher Scientific, catalog number: P330-500 )
- NP-40 10% solution (Thermo Fisher Scientific, catalog number: 28324 )
- 1 M MgCl2 (see Recipes)
- 2.5 M sucrose (see Recipes)
- TMK (see Recipes)
- TMK + 1% NP-40 (see Recipes)
- S1 buffer (see Recipes)
- S2 buffer (see Recipes)
- Cell lysis buffer (see Recipes)
Equipment
- Ventilated microcentrifuge (Fisher Scientific, model: accuSpinTM Micro 17R, catalog number: 13-100-676 )
- Gilson PIPETMAN Classic pipets (Gilson, model: P1000, catalog number: F123602 )
- Gilson PIPETMAN Classic pipets (Gilson, model: P20, catalog number: F123600 )
- Gilson PIPETMAN Classic pipets (Gilson, model: P200, catalog number: F123601 )
- Gilson PIPETMAN Classic pipets (Gilson, model: P2, catalog number: F144801 )
- Water bath (Fisher Scientific, model: FS-14 )
- Vortexer (Fisher Scientific, catalog number: 02-216-108 )
- ABI Prism 7900HT sequence detection system (PE-Applied Biosystems) (Thermo Fisher Scientific, Applied BiosystemsTM, model: ABI PRISMTM 7900HT )
Procedure
文章信息
版权信息
© 2017 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Galvis, A. E., Fisher, H. E. and Camerini, D. (2017). NP-40 Fractionation and Nucleic Acid Extraction in Mammalian Cells. Bio-protocol 7(20): e2584. DOI: 10.21769/BioProtoc.2584.
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分类
分子生物学 > DNA > DNA 提取
细胞生物学 > 细胞器分离 > 细胞核
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