Autophagy assay cell type - Proximal tubular cells (rPT)

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
	- Incubate Primary antibody at 4C overnight. - If there is high background or non specific bands dilute the secondary antibody in buffer containing 1-5% normal serum from the same species as the sample

Protocol tips
- Incubate Primary antibody at 4C overnight. - If there is high background or non specific bands dilute the secondary antibody in buffer containing 1-5% normal serum from the same species as the sample

Manufacturer protocol Publication protocol 1 Relevant paper

Anti-Beclin 1 antibody (ab62557)

Abcam

Upstream tips	Protocol tips	Downstream tips
	If there is high background or non specific bands dilute the secondary antibody in buffer containing 1-5% normal serum from the same species as the sample

Protocol tips
If there is high background or non specific bands dilute the secondary antibody in buffer containing 1-5% normal serum from the same species as the sample

Manufacturer protocol Publication protocol 1 Relevant paper

LAMP-1 Antibody (H4A3)

Santa Cruz Biotechnology

Upstream tips	Protocol tips	Downstream tips
	- Dilute at 1 : 80 diluted in PBS. - Incubate cells at 4 °C overnight,

Protocol tips
- Dilute at 1 : 80 diluted in PBS. - Incubate cells at 4 °C overnight,

Manufacturer protocol Publication protocol 1 Relevant paper

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