Autophagy assay cell type - prostate cancer PPC1 cells

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 2 matching solutions for this experiment

Anti-ATG4B antibody produced in rabbit

Sigma-Aldrich

Upstream tips	Protocol tips	Downstream tips
Lyse cells in in RIPA buffer (1% NP40, 50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.25% sodium deoxycholate, 1% sodium dodecyl sulfate (SDS), protease inhibitor cocktail and phosphatase inhibitor)	Incubate overnight at 4°C with primary antibody

Upstream tips
Lyse cells in in RIPA buffer (1% NP40, 50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.25% sodium deoxycholate, 1% sodium dodecyl sulfate (SDS), protease inhibitor cocktail and phosphatase inhibitor)
Protocol tips
Incubate overnight at 4°C with primary antibody

Manufacturer protocol Publication protocol 1 Relevant paper

Anti-LC3B antibody produced in rabbit

Sigma-Aldrich

Upstream tips	Protocol tips	Downstream tips
Lyse cells in in RIPA buffer (1% NP40, 50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.25% sodium deoxycholate, 1% sodium dodecyl sulfate (SDS), protease inhibitor cocktail and phosphatase inhibitor)	Incubate overnight at 4°C with primary antibody

Upstream tips
Lyse cells in in RIPA buffer (1% NP40, 50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.25% sodium deoxycholate, 1% sodium dodecyl sulfate (SDS), protease inhibitor cocktail and phosphatase inhibitor)
Protocol tips
Incubate overnight at 4°C with primary antibody

Manufacturer protocol Publication protocol 1 Relevant paper

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