Projected confocal z-stack of HeLa cells, untreated (left), nutrient-starved with EBSS (2 hr; middle), or treated with Chloroquine #14774 (50 μM, 24 hr; right), using LC3B (E5Q2K) Mouse mAb (Alexa Fluor® 488 Conjugate) (green). Samples were mounted in ProLong® Gold Antifade Reagent with DAPI #8961 (blue).
Confocal immunofluorescent analysis of HCT 116 cells, either untreated (left) or treated with Chloroquine #14774 (50 µM, overnight) (center), or LC3B HCT 116 knockout cells treated with Chloroquine #14774 (50 µM, overnight) (right) using LC3B (E5Q2K) Mouse mAb (Alexa Fluor® 488 Conjugate) (green). Actin filaments were labeled with β-Actin (8H10D10) Mouse mAb (red) and nuclei were labeled with DAPI #4083 (blue).
|REACTIVITY||H M R|
This Cell Signaling Technology antibody is conjugated to Alexa Fluor® 488 fluorescent dye and tested in-house for direct immunofluorescent analysis in human cells. This antibody is expected to exhibit the same species cross-reactivity as the unconjugated LC3B (E5Q2K) Mouse mAb #83506.
|Immunofluorescence (Immunocytochemistry)||1:100 - 1:200|
Supplied in PBS (pH 7.2), less than 0.1% sodium azide and 2 mg/ml BSA. Store at 4°C. Do not aliquot the antibody. Protect from light. Do not freeze.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.
NOTE: Cells should be grown, treated, fixed and stained directly in multi-well plates, chamber slides or on coverslips.
NOTE: All subsequent incubations should be carried out at room temperature unless otherwise noted in a humid light-tight box or covered dish/plate to prevent drying and fluorochrome fading.
posted December 2010
Protocol Id: 221
LC3B (E5Q2K) Mouse mAb (Alexa Fluor® 488 Conjugate) recognizes endogenous levels of total LC3B protein. This antibody detects both type I and type II forms of LC3B. Cross-reactivity was not detected with other family members.
Human, Mouse, Rat
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues near the amino terminus of human LC3B protein.
Autophagy is a catabolic process for the autophagosomic-lysosomal degradation of bulk cytoplasmic contents (1,2). Autophagy is generally activated by conditions of nutrient deprivation, but it has also been associated with a number of physiological processes including development, differentiation, neurodegenerative diseases, infection, and cancer (3). Autophagy marker Light Chain 3 (LC3) was originally identified as a subunit of microtubule-associated proteins 1A and 1B (termed MAP1LC3) (4) and subsequently found to contain similarity to the yeast protein Apg8/Aut7/Cvt5 critical for autophagy (5). Three human LC3 isoforms (LC3A, LC3B, and LC3C) undergo posttranslational modifications during autophagy (6-9). Cleavage of LC3 at the carboxy terminus immediately following synthesis yields the cytosolic LC3-I form. During autophagy, LC3-I is converted to LC3-II through lipidation by a ubiquitin-like system involving Atg7 and Atg3 that allows for LC3 to become associated with autophagic vesicles (6-10). The presence of LC3 in autophagosomes and the conversion of LC3 to the lower migrating form, LC3-II, have been used as indicators of autophagy (11).
Explore pathways + proteins related to this product.
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