LC3B antibody

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	- Use 2 µg/ml of LC3B Ab at a dilution of 1∶2000 and incubate for overnight at 4C

Protocol tips
- Use 2 µg/ml of LC3B Ab at a dilution of 1∶2000 and incubate for overnight at 4C

Publication protocol

Cell lysis, SDS-PAGE and western blotting
Cells were lysed on ice for 15 min in 1×RIPA lysis buffer. Protein concentration was measured using the Bradford assay. The amount of protein was approximately 30∼60 µg (concentration measured by Bradford method: 1.5∼3 mg/ml) per sample. Equal amounts of cell extracts were boiled for 5 min in the presence of 1% 2-mercaptoethanol and 2% sodium dodecyl sulfate (SDS). Samples were electrophoresed on 15% SDS-poly-acrylamide gels, and then transferred to polyvinylidene difluoride (PVDF) membrane. Membranes were blocked with 5% (wt/vol) milk proteins/Tris-buffered saline and incubated overnight at 4°C with the primary antibody: rabbit anti-LC3 (2 µg/ml), rabbit anti-Beclin 1 (1 µg/ml), rabbit anti-Atg7 (1 µg/ml), rabbit anti-cleaved caspase 3 (2 µg/ml), anti-phospho-P44/42 MAP Kinase (2 µg/ml), anti-P44/42 MAP Kinase (2 µg/ml), anti-SAPK/JNK (2 µg/ml), anti-phospho-SAPK/JNK (2 µg/ml), anti-p38MAP Kinase (2 µg/ml) and anti-phospho-p38 MAP Kinase (2 µg/ml). Bound antibodies (dilution:1∶2000) were detected with appropriate secondary antibodies and enhanced chemiluminescence. Same blots were probed with anti-β-Actin to monitor protein loading and transferring. A densitometric quantification of western blot signal intensity of membranes was performed.

Autophagy assays
Autophagy was examined by analyzing the formation of fluorescent puncta of autophagosomes in GFP-LC3 transfected cells and immunoblot analysis of LC3-II and Beclin 1, another essential autophagy related protein. In fluorescence microscopy experiments, NRK52E cells were transiently transfected with GFP-LC3 plasmid and treated with AAI as described above. After treatment, the cells were fixed with 4% paraformaldehyde. Autophagy was evaluated by examining the punctate forms (type II) of the autophagy marker LC3 based on GFP-LC3. Quantitation of autophagy was done based on the percentage of GFP-LC3-positive autophagic vacuoles or cells with LC3 punctate dots. Twenty fields of ×600 magnification with 20 to 30 GFP-labeled green cells per field were counted in each condition [21].

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 - NRK-52E using LC3B antibody from GeneTex.

Manufacturer protocol

Download the product protocol from GeneTex for LC3B antibody below.

Download manufacturer protocol

Videos

Check out videos that might be relevant for performing Autophagy assay cell type - NRK-52E using LC3B antibody from GeneTex. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.