This antibody specifically recognizes both forms of endogenous LC3, the cytoplasmic LC3-I (18 kDa) as well as the lipidated form generated during autophagosome and autophagolysosome formation: LC3-II (16 kDa). Immunocytochemical staining of cells with LC3 antibody Cat.-No AM20212PU-N (Clone 5F10) reveals the specific punctate distribution of endogenous LC3-II as a hallmark of autophagic activity.
Immunoblotting: 0.5 μg/mL for HRPO/ECL detectionRecommended blocking buffer: Casein/Tween 20 based blocking and blot incubationbuffer. We strongly recommend to use PVDF membranes for immunoblot analysis. Immunocytochemistry: Use at 1-10 μg/mL (Paraformaldehyd/Methanol fixation). Included Positive Control: Cell lysate from untreated Neuro 2A (See Protocols).
Protocol
Positive Control: Cell lysate from untreated Neuro 2A cells, brain endothelioma (Mouse)Format: Lyophilized cell lysate from serum starved Neuro 2A. Reconstitution: Restore by addition of 200 µl H2O. After complete solubilization add 200µl 2x SDS-PAGE sample buffer, mix and incubate at 90°C for 5 min. Application: The positive control cell lysate is recommended for immunoblot applications. 20 µl of positive control cell lysate correspond to ca. 20. 000 cells. Use 20 µl/lane (mini gel) for HRPO/ECL detection of the target proteins. Please NOTE: The lyophilized cell lysates conatin SDS and are not recommended forapplications with native proteins such as in immunoprecipitation.
Restrictions
For Research Use only
Reconstitution
Restore with 1 mL H2O (15 min, RT)
Buffer
1 mLPBS, 0.09 % Sodium Azide, PEG and Sucrose
Preservative
Sodium azide
Precaution of Use
This product contains sodium azide: a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.
Storage
-20 °C/-80 °C
Storage Comment
Store lyophilized (preferably in a desiccator) at -20 °C and reconstituted (aliquote andfreeze in liquid nitrogen) at -80 °C. Thaw aliquots at 37 °C. Thawed aliquots may be stored at 4 °C up to 3 months. Avoid repeated freezing and thawing. Shelf life: one year from despatch.
Expiry Date
12 months
Target
LC3B (MAP1LC3B)
(Microtubule-Associated Protein 1 Light Chain 3 beta (MAP1LC3B))
Autophagy is an alternative process of proteasomal degradation for some long-lived proteins or organelles. Alterations in the autophagic-lysosomal compartment have been linked to neuronal death in many neurodegenerative disorders as well as in transmissible neuronal pathologies (prion diseases). Genetic studies in yeast have shown that Autophagy-defective Gene-8 (Atg-8) represents a specific marker for autophagy. Among the four families of mammalian Atg8-related proteins only LC3 (microtubule-associated protein1 light chain 3) is expressed at sufficient high levels and efficiently recruited to autophagic vesicles in cells and tissues. During autophagy the cytoplasmic form, LC3-I is processed and recruited to autophagosomes, where LC3-II is generated by site specific proteolysis near to the C-terminus. Autophagic vacuoles have been also reported frequently in cardiomyopathies or muscle cells exposed to different experimental settings.Synonyms: Autophagy-related protein LC3 B, Autophagy-related ubiquitin-like modifier LC3 B, MAP1A / 1B light chain 3 B, MAP1A/MAP1B, MAP1LC3B, Map1alc3, Map1lc3, Map1lc3b, Microtubule-associated protein 1 light chain 3 beta, Microtubule-associated proteins 1A/1B light chain 3B