Anti-LC3 pAb

Protocol tips

Upstream tips	Protocol tips	Downstream tips
- For Western Blot, lyse cells with RIPA buffer (50 mM Tris, 1.0 mM EDTA, 150 mM NaCl, 0.1% Triton X-100, 1% sodium deoxycholate, 1 mM PMSF)	- For Western Blot dilute primary Ab at 1:1000 and incubate at 4 °C overnight. - For IF, dilute primary Ab between 1:500-:1000 and incubate at 4 °C overnight.

Upstream tips
- For Western Blot, lyse cells with RIPA buffer (50 mM Tris, 1.0 mM EDTA, 150 mM NaCl, 0.1% Triton X-100, 1% sodium deoxycholate, 1 mM PMSF)
Protocol tips
- For Western Blot dilute primary Ab at 1:1000 and incubate at 4 °C overnight. - For IF, dilute primary Ab between 1:500-:1000 and incubate at 4 °C overnight.

Publication protocol

Western blotting
Cells were lysed with RIPA buffer (50 mM Tris, 1.0 mM EDTA, 150 mM NaCl, 0.1% Triton X-100, 1% sodium deoxycholate, 1 mM PMSF). Protein concentrations were quantified by the BCA protein assay kit (Pierce, 23225). Proteins were resolved on 12% SDS-PAGE, then transferred to PVDF (Bio-Rad) membranes. After blocking, the membranes were incubated with primary antibodies at 4 °C overnight, then incubated with secondary antibodies at room temperature for 1 h. Target proteins were examined using Enhanced Chemiluminescence reagents (Millipore, WBKLS0100).

Immunofluorescence
Cells were fixed with 4% paraformaldehyde for 30 min, washed with PBS, and then incubated with 0.1% Triton X-100 for permeabilization. Cells were stained with anti-LC3B polyclonal antibody at 4 °C overnight, and subsequently with Alexa Fluor 488-conjugated goat anti-rabbit LgG (Abcam, ab150077) at 37 °C for 1 h. Nuclei were stained with DAPI for 5 min. Images were captured using a confocal laser scanning microscope (Olympus, FV3000).

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