Anti-LC3 Antibody

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	Dilute primary Ab at 1:500

Protocol tips
Dilute primary Ab at 1:500

Publication protocol

Cells (2×106) were treated with 100 nM RAD001 for 48 h at 37°C, and then with and without 2.5 nM YM155 1 h prior to RAD001 treatment. Control cells were treated with equivalent quantities of DMSO. Cells then were lysed by incubating in lysis buffer (1% Triton, 0.1 % sodium dodecyl sulfate-SDS, 150 mM NaCl, 50 mM Tris HCl pH 7.4, 2 mM EDTA) plus a protease inhibitor cocktail tablet (Roche Applied Science, Penzburg, Germany) for 30 min at 4°C. Lysates were then centrifuged at 16,000 × g for 15 min at 4°C and the supernatant was collected. The protein concentration was evaluated using the Bio-Rad Protein Concentration assay (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Protein lysate samples (50–100 µg) were separated by molecular weight on 10, 12 or 14% SDS-PAGE and then transferred onto nitrocellulose membranes. Membranes were blocked for 1 h at room temperature in 5% non-fat dry milk and then incubated with primary antibodies overnight at 4°C, washed in Tris-buffered saline with 0.1% Tween-20 and then incubated with horseradish peroxidase-conjugated anti-mouse IgG (cat. no. A4416) or anti-rabbit IgG (cat. no. A0545) (both 1:5,000; Sigma-Aldrich, St. Louis, MO, USA) for 1 h at room temperature. The filters were then developed using enhanced chemiluminescence (Super Signal West Pico Chemiluminescent Substrate; Thermo Fisher Scientific, Inc., Waltham, MA, USA) using Kodak X-Omat films (Kodak, Rochester, NY, USA). The primary antibodies used were as follows: Rabbit anti-survivin (1:1,000; cat. no. NB500-201; Novus Biologicals, Littleton, CO, USA), mouse anti-poly (ADP-ribose) polymerase 1 (PARP1; 1:500; cat. no. SC-8007; Santa Cruz Biotechnology, Dallas, TX, USA), which is able to detect both cleaved and uncleaved PARP1, mouse anti-caspase-8 (1:500; cat. no. 9746; Cell Signaling Technology, Danvers, MA, USA) which is able to detect both cleaved and uncleaved caspase-8, mouse anti-caspase-9 (1:500; cat. no. 9508; Cell Signaling Technology), mouse anti-B cell lymphoma 2 (Bcl2; 1:250; cat. no. Sc7382; BD Biosciences, Franklin Lakes, NJ, USA), rabbit anti-BCL2 associated X, apoptosis regulator (Bax; 1:250; cat. no. Sc493; BD Biosciences), rabbit anti-LC3 (1:500; cat. no. ABC232; Sigma-Aldrich, St. Louis, MO, USA), which is capable of recognizing the doublet of LC3 composed of the two single bands of LC3I and LC3II; rabbit anti-beclin-1 (1:500; cat. no. 62557; Abcam, Cambridge, UK), mouse anti-β-actin (1:750; cat. no. A5060; Sigma Aldrich, St. Louis, MO, USA).

Full paper   Login or join for free to view the full paper.

Reviews

Discussion

Papers

Check out relevant papers found by Labettor's AI that are relevant for performing Autophagy assay cell type - MCF7 using Anti-LC3 Antibody from Sigma-Aldrich.

Manufacturer protocol

Download the product protocol from Sigma-Aldrich for Anti-LC3 Antibody below.

Download manufacturer protocol

Videos

Check out videos that might be relevant for performing Autophagy assay cell type - MCF7 using Anti-LC3 Antibody from Sigma-Aldrich. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.