Autophagy ELISA Kit (LC3-II Quantitation)
Q
Colorimetric
Quantitative
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- 抗原
- LC3-II
- 检测方法
- Colorimetric
- 应用范围
- Quantification (Q)
- 原理
- Autophagy ELISA Kit (LC3-II Quantitation) is an enzyme immunoassay developed for detection and quantitation of LC3 in cell lysate samples.
- Analytical Method
- Quantitative
- 产品特性
- Autophagy ELISA Kit (LC3-II Quantitation) is an enzyme immunoassay developed for detection and quantitation of LC3 in cell lysate samples. The kit utilizes a selective permeabilization procedure to remove the cytosolic pro-LC3 and LC3-I and retain the autophagosome membrane bound LC3-II. The quantity of LC3-II in unknown lysate sample is determined by comparing its absorbance with that of a known LC3 standard curve. Each kit provides sufficient reagents to perform up to 96 assays, including standard curve and unknown samples. Processing of LC3. During autophagy, pro-LC3 undergoes C-terminal proteolysis to form LC3-I (Cytosolic). LC3-I protein conjugates to phosphatidylethanolamine (PE) and translocates to the autophagosomal membrane (LC3-II). A selective permeabilization procedure is utilized to remove cytosolic pro-LC3 /LC3-I and retain autophagosome-bound LC3-II. LC3-II is used as a specific marker of autophagy.
- 组件
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- 100X Cytosolic LC3 Removal Reagent : One 1 mL tube
- Anti-LC3 Antibody Coated Plate : One 96-well strip plate (8 x 12).
- Anti-LC3 Antibody : One 50 μL tube of anti-LC3.
- Secondary Antibody, HRP Conjugate (1000X) : One 20 μL vial.
- Assay Diluent : One 50 mL bottle.
- 10X Wash Buffer : One 100 mL bottle.
- Substrate Solution : One 12 mL amber bottle.
- Stop Solution: One 12 mL bottle.
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- 应用备注
- Optimal working dilution should be determined by the investigator.
- 试剂准备
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- 1X Cytosolic LC3 Removal Reagent: Warm tube to room temperature. FRESHLY prepare desired amount of 1X Cytosolic LC3-I Removal Reagent by diluting the provided 100X stock 1:100 in 1X PBS containing 1 mM MgCl2 and 1 mM CaCl2. For example, add 50 μL of 100X Cytosolic LC3 Removal Reagent to 5.0 mL of 1X PBS containing 1 mM MgCl2 and 1 mM CaCl2. Do not store diluted solutions.
- 1X Wash Buffer: Dilute the 10X Wash Buffer to 1X with deionized water. Stir to homogeneity.
- Anti-LC3 Antibody and Secondary Antibody: Immediately before use dilute the Anti-LC3 Antibody 1:200 and Secondary Antibody 1:1000 with Assay Diluent. Do not store diluted solutions.
- 限制
- 仅限研究用
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- 储存条件
- -20 °C,-80 °C
- 储存方法
- Upon receipt, store Recombinant LC3 Standard at -80°C, and the Anti-LC3 Antibody and Secondary Antibody at -20°C. Store all other components at 4°C.
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- 抗原
- LC3-II
- 背景
- Autophagy is a lysosomal degradation pathway for cytoplasmic material, which is activated during stress conditions such as amino acid starvation or viral infection. Mammalian cells use autophagy during short periods of starvation to degrade nonessential cellular components in order to liberate nutrients for vital biosynthetic reactions. Recent results have shown that autophagy also contributes to development, growth regulation and cancer, as well as longevity. After induction by a stress signal such as amino acid starvation, the first step in autophagy is the formation of an autophagosome. A well published autophagosome marker protein, MAP LC3, was originally identified as a microtubule associated protein and named 'microtubule-associated-protein- light-chain-3'. LC3 is a small 16-18 kDa protein that is soluble in nonstarved cells, but becomes peripherally membrane-associated during amino acid starvation. By immunoelectron microscopy, LC3 has been shown to associate to the inner and outer limiting membranes of autophagosomes, and the membrane association is mediated by a covalent conjugation to a lipid, phosphatidylethanolamine. In Western blots, two forms of LC3 are seen, LC3-I and LC3-II. LC3-I is found in the soluble fraction, and LC3-II in the pelletable membrane fraction. Both LC3-I and LC3-II are seen in nonstarved cells, but during autophagy induction the proportion of LC3-II increases.
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