自体吞噬分析法（LC3B免疫荧光法、LC3B蛋白印迹法、吖啶橙分析法）

Xin  Zhang; Qingsong  Liu

doi:10.21769/BioProtoc.1012

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Peer-reviewed

Autophagy Assays (LC3B immunofluorescence, LC3B western blot, acridine orange assay)

自体吞噬分析法（LC3B免疫荧光法、LC3B蛋白印迹法、吖啶橙分析法）

XZ Xin Zhang

QL Qingsong Liu email

发布: 2013年12月20日第3卷第24期 DOI: 10.21769/BioProtoc.1012 浏览次数: 28019

评审: Lin Fang

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参见作者原研究论文

The authors used this protocol in:

Cover of Cancer Research, featuring study using the protocol.

Apr 2013

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Abstract

Autophagy is a dynamic cellular event that is involved in the degradation of long lived proteins and organelles in cells. Biochemical methods such as western blot to measure autophagic proteins have been increasingly used in autophagy studies because it is convenient and objective. Among them, the total amount of Microtubule-associated protein light chain 3 (LC3), the mammalian homologue of the autophagy-related Atg8 in yeast, is a very useful and most commonly used tool in autophagy studies. Other methods such as electron microscopy and immunofluorescence are also available to measure autophagy. The following protocol describes three simple and commonly used protocols for measuring autophagy in cells: LC3B immunofluorescence, western blot and acridine orange assay. Although these three methods are frequently used to provide basic information about autophagy, people should keep in mind that they are not enough to give the exact details about the autophagy flux due to the complexity of this dynamic process. In addition, acridine orange assay is only a supplementary method to detect autophagy because it also has high affinity to other organelles such as lysosomes. For further investigation about a compound’s effect on autophagy flux, additional and more complicated assays are recommended. Protocols here provide a starting point for people to get a snapshot of whether a compound can affect autophagy in tissue culture cells.

Keywords: Autophagy (自噬)

LC3 (LC3)

Acridine orange (吖啶橙)

Immunofluorescence (免疫荧光)

Western blot (Western blot)

Materials and Reagents

70% ethanol
DMEM or other medium for tissue-culture cells
Bovine Serum Albumin (BSA) (Sigma-Aldrich)
Formaldehyde
100% Methanol
PBS
Rabbit antiLC3B antibody (LC3B (D11) XP^® Rabbit mAb) (Cell Signaling Technology, catalog number: 3868 )
HRP-conjugated secondary antibody
Fluorescently labeled anti-rabbit secondary antibody (Molecular Probes, Life Technologies, catalog number: A-11008 )
ProLong^® Gold Antifade Reagent with DAPI (Life Technologies, catalog number: P36935 )
M-PER lysis buffer (Pierce Antibodies, catalog number: 78501 )
5% Non-fat dry milk
cOmplete ULTRA Protease inhibitor cocktail tablets (Roche Diagnostics, catalog number: 0 5892970001 )
PhosSTOP phosphatase inhibitor cocktail tablets (Roche Diagnostics, catalog number: 0 4906837001 )
Sample buffer (Bio-Rad Laboratories, catalog number: 161-0737 )
Enhanced chemiluminescence (ECL)
Acridine orange (Sigma-Aldrich, catalog number: A8097 )
4-15% Mini-PROTEAN TGX Precast Gel (Bio-Rad Laboratories, catalog number: 456-1085 )
E64d/pepstatin A (Sigma-Aldrich, catalog number: E8640/P5318 )
TBS (see Recipes)
TBS-Tween (see Recipes)
AbDil-Tw (see Recipes)
TBS-Tx (see Recipes)
AbDil-Tx (see Recipes)
Running buffer (see Recipes)
Transfer buffer (see Recipes)

Equipment

24-well plate
12-well plate (or 6-well plate)
1.5 ml tube
Round microscope coverslips (Fisher Scientific, catalog number: 12-545-81 ) (these coverslips are ready to use and do not require extra coating procedure)
Slides
Tweezer with fine tips
Transfer pipette
Humid chamber (can be homemade from a 15 cm plate, parafilm, foil and wet kimwipes)
Vacuum pump with a liquid bottle and pipe attached to remove washing solution
Fluorescent microscope
Western Blotting apparatus (SDS-PAGE running cassette, power supply, shaker, transfer cassette, PVDF membrane, etc.)
Tissue culture apparatus (CO₂ incubator, tissue culture hood, etc.)

Software

MetaMorph

Procedure

English

中文翻译

文章信息

版权信息

如何引用

Zhang, X. and Liu, Q. (2013). Autophagy Assays (LC3B immunofluorescence, LC3B western blot, acridine orange assay). Bio-protocol 3(24): e1012. DOI: 10.21769/BioProtoc.1012.