LC3 Antibody (APG8B) (N-term)

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Lyse cells in ice-cold mammalian lysis buffer	For WB, incubate with primary antibody diluted to 2 ug/ml in total volume of 3 mL in Blocking buffer for one hour at room temperature. For IF, stain with primary antibody for 45 min at RT in 40 ml of 1%BSA-PBS by forming a drop on the coverslip

Upstream tips
Lyse cells in ice-cold mammalian lysis buffer
Protocol tips
For WB, incubate with primary antibody diluted to 2 ug/ml in total volume of 3 mL in Blocking buffer for one hour at room temperature. For IF, stain with primary antibody for 45 min at RT in 40 ml of 1%BSA-PBS by forming a drop on the coverslip

Publication protocol

Preparation of cell lysates, immunoprecipitation, immunoblot and GST-fusion protein affinity precipitation assay
Cells were rinsed once with ice-cold phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4) and lysed in ice-cold mammalian lysis buffer (40 mM HEPES [Sigma-Aldrich, H3374], pH 7.4,100 mM NaCl, 1% Triton X-100 [Sigma-Aldrich, T8787], 25 mM glycerol phosphate, 1 mM sodiumorthovanadate, 1 mM EDTA, 10 µg/ml aprotinin [Sigma-Aldrich, A1153], and 10 µg/ml leupeptin [Sigma-Aldrich, L9783]) or RIPA buffer (40 mM HEPES, pH 7.4, 1% Triton X-100, 0.5% Na-deoxylcholate [Sigma-Aldrich, D6750], 0.1% SDS [Sigma-Aldrich, L3771], 100 mM NaCl, 1 mM EDTA, 25 mM μ-glycerolphosphate, 1 mM Na-orthovanadate, 10 μg/ml leupeptin and aprotinin) as indicated. The cell lysates were cleared by centrifugation at 15,000 xg for 15 min. In immunoprecipitation, primary antibodies were added to the lysates and incubated with rotation at 4°C for 30 min, followed by adding 20 μl of the protein A-Sepharose (Sigma-Aldrich, P3391) bead slurry (1:1) into the lysates and incubating with rotation for an additional 3 h. The immunoprecipitates were washed 3 times with lysis buffer. The cell lysates or immunoprecipitated proteins were denatured by addition of SDS-PAGE sample buffer and boiled for 5 min, resolved by 8%–14% SDS-PAGE. The proteins in the gel were transferred to PVDF membranes (EMD Millipore, IPFL00010). The immunoblot was performed as described previously, using chemo-luminescence.57

GST fusion protein expression, purification and affinity precipitation assay were performed as previously described.37,57

Immunofluorescent staining
The cells were cultured in glass-bottom culture dishes with coverslips (MatTek, P35GCOL-0–10-C) to 50 to 80% confluence. After the culture medium was aspirated, the cells were rinsed with PBS twice, fixed with 3.7% paraformaldehyde at 25°C for 10 min, and permeabilized with 0.2% Triton X-100 in PBS at 25°C for 10 min. After washing with PBS, the cells were incubated with primary antibody at 8°C overnight. The cells were washed with PBS 3 times and incubated with secondary antibody conjugated with a fluorescent dye (Molecular Probes, T-6390 or T-6391) at 37°C for 1 to 2 h. After washing with PBS 3 times, fluorescent staining of the cells was visualized under a Zeiss LSM710 confocal microscope (Peabody, MA 01960) or a Nikon Eclipse TE2000 inverted fluorescent microscope (Melville, NY 11747)

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