LC3A (D50G8) XP® Rabbit mAb

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	- Dilute Primary Ab at -1:1000. - For LC3 analyses the same PVDF-blotted membranes were cut horizontally at the 38 kDa marker so that the lower half could be blotted for LC3 while the upper half could be blotted simultaneously for actin to ensure equality of protein loading and transfer across samples.

Protocol tips
- Dilute Primary Ab at -1:1000. - For LC3 analyses the same PVDF-blotted membranes were cut horizontally at the 38 kDa marker so that the lower half could be blotted for LC3 while the upper half could be blotted simultaneously for actin to ensure equality of protein loading and transfer across samples.

Publication protocol

Samples containing 25 µg protein (5 µL/sample) were electrophoresed across 4–20% precast polyacrylamide gels (BioRad, Cat# 456-8093) using pre-stained molecular weight markers (BioRad, 161-0305; 161-0324) to follow the process in real time. Generally electrophoresis employed a Tris-glycine running buffer (BioRad, 161-0732) pH 8 operating at 90V throughout the run, but for LC3 assays (25 µg protein) the voltage was reduced to 60V for the first three-quarters of the electrophoresis then increased to 90V until the lowest molecular weight marker (aprotinin, 5 kDa) approached within 2 mm of the lower edge of the gel. Gels were wet-blotted to polyvinylidine difluoride (PVDF) membranes (Millipore, ISEQ00010) for 2h at 60V, blocked overnight in 4% bovine serum albumin (BSA, Santa Cruz Biotechnology, 9048-46-8) and developed using antibodies listed in Table I (1:1000 dilution each). For LC3 analyses the same PVDF-blotted membranes were cut horizontally at the 38 kDa marker so that the lower half could be blotted for LC3 while the upper half could be blotted simultaneously for actin to ensure equality of protein loading and transfer across samples. Blots were developed with enhanced chemiluminescence reagents (Amersham, RPN2132). Antibodies used for immunoblotting are listed in Table I, and were all used at 1:1000 dilution.

mTOR or TSC2 localization relative to lysosomes was assessed using a microscopic technique involving fluorescent double-labeling of mTOR or TSC2 and the lysosomal marker LAMP2 (Sancak eta al., 2010). Cultured rat glioma cells (RG2) treated with 50 nM rapamycin and 10 µM LKE for 24 h were fixed in 4% formaldehyde (Sigma, 47608) for 30 min, permeabilized for 30 min in 0.1% Triton X-100 (Sigma, T-9284) in PBS at room temperature; blocking of nonspecific binding sites was carried out with 2% B-PBS (BSA in PBS). Immunolabeling with rabbit anti-mTOR (Santa Cruz Biotechnology; sc-8319) (1:100), or rabbit anti-tuberin (TSC2) (Santa Cruz Biotechnology; sc-893) (1:100), and rat anti-LAMP2 mAb (Abcam, ab25339) (1:100) was carried out for 2h at room temperature. The secondary antibodies used were Alexa Fluor 488 goat anti-rabbit IgG (Molecular Probes, A11008, 1:100) and Alexa Fluor 594 goat anti-rat IgG (Molecular Probes, A11007, 1:100) applied for 30 min at room temperature. An Olympus IX 71 fluorescence microscope was used for observation and image collection. Images were captured with a charge-coupled device (CCD) camera (QImaging, Canada), recorded with Slidebook 4.2 digital microscopy software, and later analyzed using MetaMorph for Olympus Basic Offline software. A minimum of four independent experiments, each including all treatments and controls, were used for assessments of TSC2 or mTOR colocalization with LAMP2+ structures. The images were acquired at 20x magnification. Colocalization of the fluorescent tags (“green” for mTOR or TSC2 and “red” for LAMP2) as overlapped (“yellow”) area was assessed. Data from analysis of 120 images from 12 slides of 4 experiments per treatment were analyzed.

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Manufacturer protocol

Download the product protocol from Cell Signaling Technology for LC3A (D50G8) XP® Rabbit mAb below.

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