Autophagy assay cell type - MEFs (mouse embryonic fibroblasts)

As autophagy is a multi-step process which includes not just the formation of autophagosomes, but most importantly, flux through the entire system, including the degradation upon fusion with lysosomes, which makes it quite challenging for detection. There are several methods for detection in mammalian cells, including immunoblotting analysis of LC3 and p62 and detection of autophagosome formation/maturation by fluorescence microscopy, Currently, there is no single “gold standard” for determining the autophagic activity that is applicable in every experimental context, hence it is recommended to go for the combined use of multiple methods to accurately assess the autophagic activity in any given biological setting.

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Found 11 matching solutions for this experiment

Protocol tips
For WB, incubate the primary Ab at overnight 4°C and for Immunofluorescence Microscopy incubate for 1h at RT
Atg12 (D88H11) Rabbit mAb

Cell Signaling Technology

Protocol tips
For Western Blotting dilute at 1:1000 and for Immunoprecipitation at 1:100.

Incubate overnight at 4°C
Atg5 (D5F5U) Rabbit mAb

Cell Signaling Technology

Protocol tips
For Western Blotting dilute at 1:1000 and for Immunoprecipitation at 1:100.

Incubate for 16 h at 4 °C
Protocol tips
For Western Blotting dilute at 1:1000 and for Immunoprecipitation at 1:100.

Incubate for 16 h at 4 °C
Atg12 Antibody (Mouse Specific) #2011

Cell Signaling Technology

Protocol tips
Dilute primary Ab at 1:100 and incubate overnight at 4 °C and 1 h at room temperature
Protocol tips
Dilute primary Ab at 1:1000 and Incubate primary antibody overnight at 4 °C
ATG5 Antibody Novus

Novus Biologicals

Protocol tips
Dilute primary Ab at 1:500 and incubate overnight at 4 °C
LC3B (D11) XP® Rabbit mAb

Cell Signaling Technology

Protocol tips
Dilute primary Ab at 1:200 and incubate overnight at 4 °C
Protocol tips
Dilute primary Ab at 1:1000 and incubate at 4˚C overnight
Protocol tips
Dilute primary Ab at 1:2000 and incubate at 4˚C overnight
Anti-p62 (SQSTM1) (Human) pAb

MBL international corporation

Protocol tips
For WB, dilute Ab at 1:100 and for ICC at 1:500
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