LC3B Antibody

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Lyse cellls in ice cold RIPA buffer containing 1% of a protease inhibitor cocktail.	Dilur primary Ab at 1:1000 and incubate overnight at 4°C

Upstream tips
Lyse cellls in ice cold RIPA buffer containing 1% of a protease inhibitor cocktail.
Protocol tips
Dilur primary Ab at 1:1000 and incubate overnight at 4°C

Publication protocol

Western blot analysis was performed as previously described [15]. The left cerebral cortical tissue was lysed in ice cold RIPA buffer containing 1% of a protease inhibitor cocktail. The protein content of the lysate was determined by the Bradford method. Equal amounts of proteins were resolved on a 10%-12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred onto polyvinylidene fluoride (PVDF) membrane (Immobilon-P, Millipore Billerica MA, USA). The membrane was blocked with 5% non-fat dry milk in TBST (Tris-buffered saline with 0.05% Tween 20) for 2 h at room temperature, and then incubated overnight at 4°C, separately with the appropriate primary antibodies against the specific proteins. Specifically, LC3 at 1:1000 (#NB600-1384, Novus Biologicals, Minneapolis, MN, USA), Beclin-1 at 1:1000 (#NB500-249, Novus Biologicals), caspase-3 at 1:1000 (#9662, Cell Signaling Technology, Danvers, MA, USA), Bcl-2 at 1:1000 (#4223, Cell Signaling Technology), COX IV at 1:1000 (#11967, Cell Signaling Technology), Bax at 1:200 (#ab32503, Abcam, Cambridge, UK), cytochrome c at 1:10000 (#ab133504, Abcam), and β-actin at 1:5000 (Bioworld Technology, Saint Louis Park, MN, USA) in a blocking buffer. Afterwards, the membrane was washed three times with TBST for 15 min, and then incubated with the secondary antibodies, namely HRP conjugated secondary antibodies at 1:5000 (goat; Bioworld Technology) for 2 h at room temperature. Finally, following a 20-min wash with TBST, the protein bands were visualized via enhanced chemiluminescence (ECL) (Millipore, Billerica, MA, USA) and exposure to X-ray film. The western blot results were analyzed using Un-Scan-It 6.1 software (Silk Scientific Inc., Orem, UT, USA).

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