I am working on autophagy, I have seen the downrwgulaion and upregulation of autophagy by LC3B using Western Blot. When I tried to show this using eGFP-LC3 transfection, whole cell fluoresce, and no puncta like dots appears. These puncta are suppose to be the autophagosomes. Fluorescence is uniform in whole cell.
Dear Ibrar,
did you use a plasmid that was used for this purpose before? If not, it might be a problem of the Fusion (e.g. that eGFP is cleaved from LC3?). Did you include a linker between LC3 and eGFP? Unfortunately, I'm not very familiar with autophagy, but is it possible, that the pH of the autophagosomes is lower than that one of the cytosol? This could make problems with the fluorescent protein or with the linker (if I remember it right, this was at least true for lysosomes). Did you check how other people did this eGFP-LC3-fusion (papers?)?
So maybe you can describe how you did the Fusion protein (Linker?) and maybe also the promoter you use to express the fluorescent protein. Maybe the promoter is too strong?
And did you take care that the addition of this group to make LC3B out of LC3A is still possible with the fusion to eGFP?
Did you perform the positive control experiment such as HBSS starvation for more than 6-12 hours?
Why not also perform WB to detect exogeneous eGFP fused with LC3?
Dear Ibrar
As mentioned above by Nadja, I'll also guess that the problem might be at the fusion of GFP or the pH in the compartment. With the fusion you might be scavenging domains important for the sorting of the protein so you could try N-terminal and C-terminal fusions together with the use of a linker sequence. Nevertheless in this particular case the pH of the compartment might be impairing the fluorescence. You can overcome this using vectors with GFP sensitive to pH. They change spectrum and intensity in acidic environments.
Good luck!
https://www.google.com.ar/url?sa=t&source=web&rct=j&url=http://www.horizonpress.com/mbt/v/v2/01.pdf&ved=0ahUKEwj8r9zHvPLPAhUDkZAKHTMCCpsQFggpMAI&usg=AFQjCNGjczj47BavN8KQhGqQcESNJNpptw&sig2=tgo9W1HyRfrsfxuiTuf-Vg
1. Check your transfection by A) WB for GFP or B) Flow cytometry in the green channal for GFP compared to a non-transfected sample.
2. Add a positive control (either rapamycin or starvation medium).
3. Always when working in vitro add a late autophagy inhibitor (bafilomycin A1 or chloroquin) otherwise your signal might be lost for pH dependent reasons (see the explanation of Nadja above).
Success with your experiments.
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