MAP LC3β Antibody (G-2)

Protocol tips

Upstream tips	Protocol tips	Downstream tips
- This monoclonal antibody is more specific, provides a stronger signal and eliminates lot to lot variation characteristic of most, if not all, polyclonal antibodies	- Incubate Primary AB 1:1,000. at overnight at 4°C

Upstream tips
- This monoclonal antibody is more specific, provides a stronger signal and eliminates lot to lot variation characteristic of most, if not all, polyclonal antibodies
Protocol tips
- Incubate Primary AB 1:1,000. at overnight at 4°C

Publication protocol

AR42J cells were homogenized in the protein lysis buffer, and the debris was removed by centrifugation at 12,000 × g for 10 min at 4°C. The bicinchoninic acid assay (Thermo Fisher Scientific, Inc.) was used to detect protein concentration according to the protocol of Walker (17). Proteins (50 µg/lane) were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto Invitrogen polyvinylidene fluoride membranes (Thermo Fisher Scientific, Inc.). The membranes were then washed with Tris-buffered saline (10 mM Tris and 150 mM NaCl) containing 0.05% Tween-20 (TBST; Sigma-Aldrich) and blocked with 3% bovine serum albumin (BSA; Sigma-Aldrich) for 1 h on an orbital shaker at room temperature. Rabbit poly-clonal beclin-1 (1:1,000; sc-11427), LC3-II (1:1,000; sc-28266) and GAPDH (1:1,000; sc-25778) antibodies were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Goat anti-rabbit IgG horseradish peroxidase-conjugated secondary antibodies (1:2,000; ZDR-5306) were purchased from ZSGB Biotechnology (Beijing, China). TBST with 3% BSA was used to dilute the antibodies. Membranes were incubated with primary antibodies overnight at 4°C then washed in TBST for 30 min 3 times. Following washes, membranes were incubated with the secondary antibodies for 2 h at room temperature, and washed again in TBST for 30 min 3 times. Proteins were visualized by enhanced chemiluminescence (ECL), with an Amersham ECL Plus kit (GE Healthcare Life Sciences, Chalfont, UK). After mixing 1 ml ECL reagents A and B, the membrane was incubated with the mixing ECL for 2 min, according to the manufacturer's instructions. GAPDH protein levels were used as a loading control. X-ray film (Thermo Fisher Scientific, Inc.) and a Medical Film Processor (AFP Imaging Corporation, Mount Kisco, NY, USA) were used to develop protein bands in the dark. After using an Epson Perfection 3490 Photo scanner (Epson America, Inc, Plainfield, IN, USA) to obtain the TIF images of the protein bands, Image J software, version 1.49 (imagej.nih.gov/ij/) was used to quantify the expression. The time point of maximum autophagy was investigated for the following miRNA experiments.

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Manufacturer protocol

Download the product protocol from Santa Cruz Biotechnology for MAP LC3β Antibody (G-2) below.

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