LC3B (D11) XP® Rabbit mAb

Protocol tips

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	Primary Ab dilution-1:1000 dilution

Protocol tips
Primary Ab dilution-1:1000 dilution

Publication protocol

Protein lysates were extracted from Nthy-ori 3-1 cells using a whole-cell extraction kit (Merck Millipore, Billerica, MA, USA). Protein concentration was determined using a BCA protein concentration kit (Beyotime, Shanghai, China). The 5 × loading buffer (Solarbio, Beijing, China) was added, and simples were heated at 100°C for 10 min. First, 5 μg of protein was subjected to electrophoresis on a 10%–15% SDS-PAGE gel and then transferred onto a polyvinylidene difluoride (PVDF) membrane (Merck Millipore, Billerica, MA, USA). After saturation with 5% non-fat powdered milk in PBS-Tween 0.1% (PBST) (blocking buffer) for 1 h at room temperature, the membranes were incubated with primary antibodies (1:1,000 in PBST containing 2% BSA) overnight at 4°C. β-actin was used as a loading control. Following washing three times with PBST for 10 min at room temperature, membranes were probed with peroxidase-conjugated secondary antibody (anti-rabbit IgG whole-molecule diluted 1:5,000 in PBST) for 1 h at room temperature. After three washings, the signal was detected using the Pierce ECL-plus substrate (Thermo Fisher Scientific, Waltham, MA, USA) and scanned by a Fluor Chem FC3 camera system (Protein-Simple, California, USA). Images were analysed by densitometry using Alpha View software (AIC, California, USA). The results of quantitative analyses are presented graphically. Values were normalized to the loading control.

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Manufacturer protocol

Download the product protocol from Cell Signaling Technology for LC3B (D11) XP® Rabbit mAb below.

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