Autophagy assay cell type - MCF-7

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
	The silenced cells were treated with or without the autophagy inhibitors BafA1 (Millipore, 196000) or CQ (Sigma-Aldrich, C6628) and then lysed 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). The proteins were separated by SDS-PAGE and transferred onto nitrocellulose membranes for immunoblotting.

Protocol tips
The silenced cells were treated with or without the autophagy inhibitors BafA1 (Millipore, 196000) or CQ (Sigma-Aldrich, C6628) and then lysed 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). The proteins were separated by SDS-PAGE and transferred onto nitrocellulose membranes for immunoblotting.

Protocol tips

The silenced cells were treated with or without the autophagy inhibitors BafA1 (Millipore, 196000) or CQ (Sigma-Aldrich, C6628) and then lysed 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). The proteins were separated by SDS-PAGE and transferred onto nitrocellulose membranes for immunoblotting.

Manufacturer protocol Publication protocol 1 Relevant paper

Anti-LC3B antibody produced in rabbit

Sigma-Aldrich

Upstream tips	Protocol tips	Downstream tips
	The silenced cells were treated with or without the autophagy inhibitors BafA1 (Millipore, 196000) or CQ (Sigma-Aldrich, C6628) and then lysed 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). The proteins were separated by SDS-PAGE and transferred onto nitrocellulose membranes for immunoblotting.

Protocol tips
The silenced cells were treated with or without the autophagy inhibitors BafA1 (Millipore, 196000) or CQ (Sigma-Aldrich, C6628) and then lysed 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). The proteins were separated by SDS-PAGE and transferred onto nitrocellulose membranes for immunoblotting.

Protocol tips

Manufacturer protocol Publication protocol 1 Relevant paper

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