Autophagy assay cell type - NRK

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 3 matching solutions for this experiment

Upstream tips
- Lysis can be performed at 4°C in lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100) supplemented with a cocktail of protease inhibitors
Protocol tips
- Incubate primary and secondary antibodies for 1 h at room temperature, or overnight at 4°C.
Anti-LC3 pAb

MBL international corporation

Protocol tips
- Stain with 10 µg/ml of antibody in blocking buffer for 1 h.
Protocol tips
- Dilute Primary Ab at-1:200 dilution
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