Autophagy assay cell type - AR42J

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

Anti-LC3B antibody produced in rabbit

Sigma-Aldrich

Upstream tips	Protocol tips	Downstream tips
	Western blots were performed to determine the expression levels of LC3, p62 and β-actin. Briefly, AR42J cells were homogenized in T-per buffer (Thermo Fisher) with a protease inhibitor cocktail (Thermo Fisher). Lysates were centrifuged at 16,000 g for 15 min, and then, the samples were boiled in loading buffer before western blot analysis. The protein content was determined by the BCA assay. The western blot was performed according to standard procedures. The protein bands were detected by a ChemiDOC XRS imaging system (Bio-Rad, USA), and β-actin served as a loading control.

Protocol tips
Western blots were performed to determine the expression levels of LC3, p62 and β-actin. Briefly, AR42J cells were homogenized in T-per buffer (Thermo Fisher) with a protease inhibitor cocktail (Thermo Fisher). Lysates were centrifuged at 16,000 g for 15 min, and then, the samples were boiled in loading buffer before western blot analysis. The protein content was determined by the BCA assay. The western blot was performed according to standard procedures. The protein bands were detected by a ChemiDOC XRS imaging system (Bio-Rad, USA), and β-actin served as a loading control.

Protocol tips

Western blots were performed to determine the expression levels of LC3, p62 and β-actin. Briefly, AR42J cells were homogenized in T-per buffer (Thermo Fisher) with a protease inhibitor cocktail (Thermo Fisher). Lysates were centrifuged at 16,000 g for 15 min, and then, the samples were boiled in loading buffer before western blot analysis. The protein content was determined by the BCA assay. The western blot was performed according to standard procedures. The protein bands were detected by a ChemiDOC XRS imaging system (Bio-Rad, USA), and β-actin served as a loading control.

Manufacturer protocol Publication protocol 1 Relevant paper

P62/SQSTM1 Antibody

Proteintech Group

Upstream tips	Protocol tips	Downstream tips
- Tris-Tricine gels separate low MW proteins(less than 20 kDa) beer than Tris-Glycine gels. Gradient polyacrylamide gels can provide sharper bands and they separate a broader range of MW sizes on one gel, such as 10-500 kDa.	- Incubate primary and secondary antibodies for two hours at room temperature

Upstream tips
- Tris-Tricine gels separate low MW proteins(less than 20 kDa) beer than Tris-Glycine gels. Gradient polyacrylamide gels can provide sharper bands and they separate a broader range of MW sizes on one gel, such as 10-500 kDa.
Protocol tips
- Incubate primary and secondary antibodies for two hours at room temperature

Manufacturer protocol Publication protocol 1 Relevant paper

MAP LC3β Antibody (G-2)

Santa Cruz Biotechnology

Upstream tips	Protocol tips	Downstream tips
- This monoclonal antibody is more specific, provides a stronger signal and eliminates lot to lot variation characteristic of most, if not all, polyclonal antibodies	- Incubate Primary AB 1:1,000. at overnight at 4°C

Upstream tips
- This monoclonal antibody is more specific, provides a stronger signal and eliminates lot to lot variation characteristic of most, if not all, polyclonal antibodies
Protocol tips
- Incubate Primary AB 1:1,000. at overnight at 4°C

Manufacturer protocol Publication protocol 1 Relevant paper

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