Cell Signaling Technology
LC3B Antibody
2775
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Upon induction of autophagy LC3B-I is converted to LC3B-II through lipidation. This conversion is used to indicate the induction of autophagy under various conditions like starvation or treatments. The autophagy dependent degradation of a protein was an interest and upon treatment of 2 Deoxy Glucose (which is known inducer of autophagy) the induction of autophagy in the cells was confirmed by increase in LC3B-II.
Western Blot
HeLa cell lysate -50ug total protein
Primary antibody (1:1000) in dilution buffer (TBST with 5% BSA) with gentle agitation overnight at 4°C.
TBST with 5% w/v nonfat dry milk
Incubated the membrane with the Goat Anti-Rabbit IgG, H & L Chain Specific Peroxidase Conjugate (Cat No: 401315) 1:10000 diluted in 10 ml of blocking buffer with gentle agitation for 1 hr at room temperature.
None
Incubated the membrane with appropriate amount of Luminata Crescendo Western HRP substrate (WBLUR0100 Merckmillipore) for 5 min at room temperature. Drained the membrane of excess developing solution (without letting it to dry) wrapped in transparent plastic sheet and exposed the x-ray film for 1 min.
Up on treatment of 2 Deoxy glucose there was an increase in LC3B-II confirming the induction of autophagy inthe cells. LC3B antibody detects both LC3B-I and LC3B-II.
The westen blotting should be done using PVDF membranes rather than nitrocellulose membranes
It works well as described
A good reagent