Anti-LC3B antibody produced in rabbit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Lyse cells in RIPA buffer (PBS, pH 7.4, 1.0% IPEGAL CA630; 0.5% sodium deoxycholate; 0.1% SDS containing freshly added proteinase inhibitors	Primary antibody dilution 1:750

Upstream tips
Lyse cells in RIPA buffer (PBS, pH 7.4, 1.0% IPEGAL CA630; 0.5% sodium deoxycholate; 0.1% SDS containing freshly added proteinase inhibitors
Protocol tips
Primary antibody dilution 1:750

Publication protocol

Cells were lysed in a modified RIPA buffer (PBS, pH 7.4, 1.0% IPEGAL CA630 [Sigma-Aldrich, I8896]; 0.5% sodium deoxycholate [Sigma-Aldrich, D6750]; 0.1% SDS [Fisher, BP166]) containing freshly added proteinase inhibitors [Roche, 04693116001]). ATG5, ATG14, SQSTM1, and actin were resolved by electrophoresis in a 7.5% Tris-HCl polyacrylamide gel, while a 15% polyacrylamide gel (Bio-Rad Criterion, 345-0019) was used to resolve LC3 and its 2 isoforms LC3-I and LC3-II. Proteins were transferred to polyvinylidene difluoride (PVDF) membranes then blocked in tris-buffered saline (NaCl 137 mM, KCl 2.7 mM, Tris-Cl 25 mM, pH 7.4) containing 0.2% polysorbate (Tween 20; Fisher, BP337) detergent and 5% powdered milk. Membranes were then incubated in this blocking buffer with primary antibodies (and dilutions) including: LC3 (1:750; Sigma-Aldrich, L7543), ATG5 (1:1000; Sigma-Aldrich, A0856), ATG14 (1:500; Cell Signaling Technology, 5504), SQSTM1 (1:1000; Santa Cruz Biotechnology, sc-28359) and pan-actin antibody against all 6 isoforms of actin (1:2000; Millipore, MAB1501R). Primary antibodies were detected by horseradish peroxidase–labeled secondary antibody (Goat anti-rabbit, Pierce 31460; Goat anti-mouse, Pierce, 31430) binding, which was detected using enhanced chemiluminescence.

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