Anti-LC3B antibody produced in rabbit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
- Lysis can be performed at 4°C in lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100) supplemented with a cocktail of protease inhibitors	- Incubate primary and secondary antibodies for 1 h at room temperature, or overnight at 4°C.

Upstream tips
- Lysis can be performed at 4°C in lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100) supplemented with a cocktail of protease inhibitors
Protocol tips
- Incubate primary and secondary antibodies for 1 h at room temperature, or overnight at 4°C.

Publication protocol

Cells were washed in cold PBS and subjected to lysis at 4°C in lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100) supplemented with a cocktail of protease inhibitors (Sigma-Aldrich). Lysates were cleared by centrifugation at 16,000 x g for 20 min, and protein concentration determined with a protein assay solution (Bio-Rad Laboratories, Hercules, CA, USA). Samples with equivalent amount of protein were boiled for 5 min with Laemmli SDS-PAGE sample buffer, and analyzed by SDS-PAGE. Proteins were electroblotted onto nitrocellulose membranes, and membranes were incubated sequentially with primary and secondary antibodies for 1 h at room temperature, or overnight at 4°C. Chemiluminescence protein detection was performed using Pierce Western Blotting Substrate (Thermo Scientific). As an internal loading control, β-actin levels were examined on the same nitrocellulose membranes. Quantification of chemiluminiscence signal was carried out using UN-SCAN-IT software (Silk Scientific Corporation, Orem, UT, USA).

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Manufacturer protocol

Download the product protocol from Sigma-Aldrich for Anti-LC3B antibody produced in rabbit below.

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