Anti-LC3-I/II Antibody

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	- incubate primary Ab with agitation for 1 hour.	- If high background or Non-specific bands is present,Try different blocking strategies: Longer blocking times, higher % of blocker, a different blocker, inclusion of blocker protein in antibody dilutions or try a fresh batch of the same blocker.

Protocol tips
- incubate primary Ab with agitation for 1 hour.
Downstream tips
- If high background or Non-specific bands is present,Try different blocking strategies: Longer blocking times, higher % of blocker, a different blocker, inclusion of blocker protein in antibody dilutions or try a fresh batch of the same blocker.

Publication protocol

Western blotting
After treatment of A375 and C8161 cells with curcumin (25 and 15 µM, respectively) for 24 h, the cells were harvested and incubated in lysis buffer on ice for 30 min. Then the lysate was clarified by centrifugation at 12,000 x g for 10 min at 4°C to obtain the supernatant (total cell lysate). The total protein concentration was determined using the Coomassie brilliant blue (CBB) method. For western blotting, 25 µg of total protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membranes. After blocking of non-specific binding sites with 5% non-fat dry milk for 2 h at room temperature, membranes were incubated with respective primary antibodies at appropriate concentrations overnight at 4°C. After washing the membranes to remove unbound primary antibodies, they were incubated with either horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibody (1:5,000) for 1 h at room temperature. Finally the membranes were washed with PBS and chemiluminescence developed using ECL kit (APG BIO, Shanghai, China) for 1 min. Protein bands were visualized by image scanning and the optical density for each band was measured using Image Lab software (version 4.0; Bio-Rad, USA) after data were normalized to β-actin as an internal control.

Transfection and fluorescence microscopy:
Cells were transfected with a plasmid expressing GFP-LC3 using Lipofectamine 2000 according to the manufacturer's instructions. After 16 h post-transfection with GFP-LC3, A375 and C8161 cells were incubated with curcumin (25 and 15 µM, respectively) or rapamycin (100 nM, used as a positive control) or DMSO at 37°C for 24 h. Dot formation by GFP-LC3 was detected under a fluorescence microscope (DP73; Olympus, Japan) following drug treatment. Transfected cells were considered to have accumulated autophagosomes when five or more puncta were observed. A total of 100 transfected cells were examined/well, and the percentage of cells showing autophagy were counted in triplicate. The experiment was independently repeated three times.

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