LC3A/B (D3U4C) XP® Rabbit mAb

Protocol tips

Upstream tips	Protocol tips	Downstream tips
- Lyse cells in Laemmli buffer containing 60 mM Tris-HCl pH 6.8, 2% (w/v) SDS, 10 mM EDTA, 10% (w/v) glycerol	- Dilute Primary Ab at -1:1000. - Incubate Ab for overnight at 4°C

Upstream tips
- Lyse cells in Laemmli buffer containing 60 mM Tris-HCl pH 6.8, 2% (w/v) SDS, 10 mM EDTA, 10% (w/v) glycerol
Protocol tips
- Dilute Primary Ab at -1:1000. - Incubate Ab for overnight at 4°C

Publication protocol

In parallel with autophagic vacuoles analysis, OLE-treated cells were analyzed by western blotting. To this purpose, the cells were lysed directly in Laemmli buffer (60 mM Tris-HCl pH 6.8, 2% (w/v) SDS, 10 mM EDTA, 10% (w/v) glycerol). Before immunoblotting, protein concentration was determined with a BCA detection kit (Pierce, USA) and adjusted to equal concentrations across different samples. The samples were added with β-mercaptoethanol and bromophenol blue, boiled for 10 min, clarified at 10000 × g for 10 min, run on 12% SDS-PAGE and transferred to PVDF membranes (Amersham Bioscience, UK). After blocking with 5.0% (w/v) BSA in 0.1% (v/v) PBS-Tween-20 or directly with methanol, the membranes were incubated overnight at 4°C with specific primary antibodies: rabbit polyclonal anti-beclin1 antibody (1:2000, Abcam), rabbit polyclonal anti-LC3II A/B antibody (1:1000, Cell Signaling), rabbit monoclonal anti-phospho-AMPKα (Thr172) and anti-AMPKα antibodies (1:1000, Cell Signaling). Mouse monoclonal anti-β−actin antibody (1:1000, Santa Cruz Biotechnology Inc.) was used for protein load normalization. The day after, the blots were incubated for 1.0 h with specific secondary antibodies (1:10000, goat anti-rabbit antibody and goat anti-mouse antibody, Molecular Probes, Life Technologies); the immunoreactive bands were detected with the Immobilon Western Chemiluminescent HRP substrate (Millipore) or SuperSignal West Pico chemiluminescent substrate (Thermo Fisher Scientific) and quantified by densitometric analysis using a ChemiDoc system and the Quantity One software (Bio-Rad Laboratories, Italy). Statistical analysis of the bands was performed on results from at least three independent experiments, using ANOVA and Bonferroni's post-test.

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 - RIN-5F using LC3A/B (D3U4C) XP® Rabbit mAb from Cell Signaling Technology.

Manufacturer protocol

Download the product protocol from Cell Signaling Technology for LC3A/B (D3U4C) XP® Rabbit mAb below.

Download manufacturer protocol

Videos

Check out videos that might be relevant for performing Autophagy assay cell type - RIN-5F using LC3A/B (D3U4C) XP® Rabbit mAb from Cell Signaling Technology. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.