Introduction

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-proteinlight-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. Cell Biolabs' 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 (Figure 1). 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.

Related Products

1. CBA-401: GFP-LC3 Expression Vector

2. CBA-5115: Autophagy Immunoblot Kit (LC3-II Detection)

3. CBA-5117: Cellular Autophagy ELISA Kit (LC3-II Quantitation)

Kit Components

Box 1 (shipped at room temperature)

1. Anti-LC3 Antibody Coated Plate (Part No. 51161B): One 96-well strip plate (8 x 12).

2. 100X Cytosolic LC3 Removal Reagent (Part No. 51151A): One 1 mL tube

3. Anti-LC3 Antibody (Part No. 51162C): One 50 µL tube of anti-LC3.

4. Secondary Antibody, HRP Conjugate (1000X) (Part No. 230003): One 20 µL vial.

5. Assay Diluent (Part No. 310804): One 50 mL bottle.

6. 10X Wash Buffer (Part No. 310806): One 100 mL bottle.

7. Substrate Solution (Part No. 310807): One 12 mL amber bottle.

8. Stop Solution (Part. No. 310808): One 12 mL bottle.

Box 2 (shipped on blue ice packs)

1. Recombinant LC3 Standard (Part No. 51163D): One 100 µL tube of 1.0 μg/mL recombinant LC3 in TBS plus BSA.