Autophagy assay cell type - HEK 293

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 10 matching solutions for this experiment

GABARAP (E1J4E) Rabbit mAb

Cell Signaling Technology

Upstream tips	Protocol tips	Downstream tips
	U2OS cells were lysed in 10 mM KPO4, 1 mM EDTA, 10 mM MgCl2, 5 mM EGTA, 50 mM bis-glycerophosphate, 0.5% NP40, 0.1% Brij35, 0.1% sodium deoxycholate, 1 mM NaVO4, 5 mM NaF, 2 mM DTT, and complete protease inhibitors (Sigma, P8340-5ML). Proteins were resolved by SDS-PAGE, transferred to PVDF (for low molecular weight proteins like LC3) or nitrocellulose membranes, blocked with 4% w/v non-fat dry milk in 1x TBS-T (TBS with 0.05% Tween 20), and probed with primary antibodies in blocking buffer at 4°C overnight followed by secondary antibodies in blocking buffer for 45 minutes at room temperature. Proteins were detected by chemiluminescence using ECL (100 nM Tris, pH 6.8, luminol (1.25 mM), p-coumaric acid (198 μM), H2O2 (0.009%)) or SuperSignal West Femto maximum sensitivity substrate (Thermo Fisher, 34095).

Protocol tips
U2OS cells were lysed in 10 mM KPO4, 1 mM EDTA, 10 mM MgCl2, 5 mM EGTA, 50 mM bis-glycerophosphate, 0.5% NP40, 0.1% Brij35, 0.1% sodium deoxycholate, 1 mM NaVO4, 5 mM NaF, 2 mM DTT, and complete protease inhibitors (Sigma, P8340-5ML). Proteins were resolved by SDS-PAGE, transferred to PVDF (for low molecular weight proteins like LC3) or nitrocellulose membranes, blocked with 4% w/v non-fat dry milk in 1x TBS-T (TBS with 0.05% Tween 20), and probed with primary antibodies in blocking buffer at 4°C overnight followed by secondary antibodies in blocking buffer for 45 minutes at room temperature. Proteins were detected by chemiluminescence using ECL (100 nM Tris, pH 6.8, luminol (1.25 mM), p-coumaric acid (198 μM), H2O2 (0.009%)) or SuperSignal West Femto maximum sensitivity substrate (Thermo Fisher, 34095).

Protocol tips

U2OS cells were lysed in 10 mM KPO4, 1 mM EDTA, 10 mM MgCl2, 5 mM EGTA, 50 mM bis-glycerophosphate, 0.5% NP40, 0.1% Brij35, 0.1% sodium deoxycholate, 1 mM NaVO4, 5 mM NaF, 2 mM DTT, and complete protease inhibitors (Sigma, P8340-5ML). Proteins were resolved by SDS-PAGE, transferred to PVDF (for low molecular weight proteins like LC3) or nitrocellulose membranes, blocked with 4% w/v non-fat dry milk in 1x TBS-T (TBS with 0.05% Tween 20), and probed with primary antibodies in blocking buffer at 4°C overnight followed by secondary antibodies in blocking buffer for 45 minutes at room temperature. Proteins were detected by chemiluminescence using ECL (100 nM Tris, pH 6.8, luminol (1.25 mM), p-coumaric acid (198 μM), H2O2 (0.009%)) or SuperSignal West Femto maximum sensitivity substrate (Thermo Fisher, 34095).

Manufacturer protocol Publication protocol 1 Relevant paper

Atg16L1 (D6D5) Rabbit mAb

Cell Signaling Technology

Upstream tips	Protocol tips	Downstream tips
	U2OS cells were lysed in 10 mM KPO, 1 mM EDTA, 10 mM MgCl, 5 mM EGTA, 50 mM bis-glycerophosphate, 0.5% NP40, 0.1% Brij35, 0.1% sodium deoxycholate, 1 mM NaVO, 5 mM NaF, 2 mM DTT, and complete protease inhibitors (Sigma, P8340-5ML). Proteins were resolved by SDS-PAGE, transferred to PVDF (for low molecular weight proteins like LC3) or nitrocellulose membranes, blocked with 4% w/v non-fat dry milk in 1x TBS-T (TBS with 0.05% Tween 20), and probed with primary antibodies in blocking buffer at 4°C overnight followed by secondary antibodies in blocking buffer for 45 minutes at room temperature. Proteins were detected by chemiluminescence using ECL (100 nM Tris, pH 6.8, luminol (1.25 mM), p-coumaric acid (198 μM), HO (0.009%)) or SuperSignal West Femto maximum sensitivity substrate (Thermo Fisher, 34095).

Protocol tips
U2OS cells were lysed in 10 mM KPO, 1 mM EDTA, 10 mM MgCl, 5 mM EGTA, 50 mM bis-glycerophosphate, 0.5% NP40, 0.1% Brij35, 0.1% sodium deoxycholate, 1 mM NaVO, 5 mM NaF, 2 mM DTT, and complete protease inhibitors (Sigma, P8340-5ML). Proteins were resolved by SDS-PAGE, transferred to PVDF (for low molecular weight proteins like LC3) or nitrocellulose membranes, blocked with 4% w/v non-fat dry milk in 1x TBS-T (TBS with 0.05% Tween 20), and probed with primary antibodies in blocking buffer at 4°C overnight followed by secondary antibodies in blocking buffer for 45 minutes at room temperature. Proteins were detected by chemiluminescence using ECL (100 nM Tris, pH 6.8, luminol (1.25 mM), p-coumaric acid (198 μM), HO (0.009%)) or SuperSignal West Femto maximum sensitivity substrate (Thermo Fisher, 34095).

Protocol tips

U2OS cells were lysed in 10 mM KPO, 1 mM EDTA, 10 mM MgCl, 5 mM EGTA, 50 mM bis-glycerophosphate, 0.5% NP40, 0.1% Brij35, 0.1% sodium deoxycholate, 1 mM NaVO, 5 mM NaF, 2 mM DTT, and complete protease inhibitors (Sigma, P8340-5ML). Proteins were resolved by SDS-PAGE, transferred to PVDF (for low molecular weight proteins like LC3) or nitrocellulose membranes, blocked with 4% w/v non-fat dry milk in 1x TBS-T (TBS with 0.05% Tween 20), and probed with primary antibodies in blocking buffer at 4°C overnight followed by secondary antibodies in blocking buffer for 45 minutes at room temperature. Proteins were detected by chemiluminescence using ECL (100 nM Tris, pH 6.8, luminol (1.25 mM), p-coumaric acid (198 μM), HO (0.009%)) or SuperSignal West Femto maximum sensitivity substrate (Thermo Fisher, 34095).

Manufacturer protocol Publication protocol 1 Relevant paper

CYTO-ID® Autophagy detection kit

Enzo Life Sciences

Upstream tips	Protocol tips	Downstream tips
This kit uses a green fluorescent probe that reacts with autophagic vacuoles and is detectable by flow cytometry		Incase of Low CYTO-ID Green dye staining in all treatments, increase the reagent concentration (500X dilution of the dye is recommended) and the incubation time. Incase of High CYTO-ID Green staining, it could be that the cell culture medium was depleted of nutrients and so change media 4-8 hours before the experiment

Upstream tips
This kit uses a green fluorescent probe that reacts with autophagic vacuoles and is detectable by flow cytometry
Downstream tips
Incase of Low CYTO-ID Green dye staining in all treatments, increase the reagent concentration (500X dilution of the dye is recommended) and the incubation time. Incase of High CYTO-ID Green staining, it could be that the cell culture medium was depleted of nutrients and so change media 4-8 hours before the experiment

Manufacturer protocol Publication protocol 1 Relevant paper

Notch 1 Antibody (C-20)

Santa Cruz Biotechnology

Upstream tips	Protocol tips	Downstream tips
	Dilutions:immunoprecipitation [1-2 µg per 100-500 µg of total protein (1 ml of cell lysate)], immunofluorescence (starting dilution 1:50, dilution range 1:50-1:500), For immunoprecipitation, incubate cells for overnight after Ab treatment. For Immunofluorescence staining, incubate cells for 1 hr

Protocol tips
Dilutions:immunoprecipitation [1-2 µg per 100-500 µg of total protein (1 ml of cell lysate)], immunofluorescence (starting dilution 1:50, dilution range 1:50-1:500), For immunoprecipitation, incubate cells for overnight after Ab treatment. For Immunofluorescence staining, incubate cells for 1 hr

Manufacturer protocol Publication protocol 1 Relevant paper

BECN1 Antibody (H-300)

Santa Cruz Biotechnology

Upstream tips	Protocol tips	Downstream tips
Lyse cells inradioimmunoprecipitation assay buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM PMSF, 1 mM DTT, and 2 μg/ml each of leupeptin and aprotinin]	Dilute primary Ab at 1:200

Upstream tips
Lyse cells inradioimmunoprecipitation assay buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM PMSF, 1 mM DTT, and 2 μg/ml each of leupeptin and aprotinin]
Protocol tips
Dilute primary Ab at 1:200

Manufacturer protocol Publication protocol 1 Relevant paper

LC3B Rabbit Antibody

Cell Signaling Technology

Upstream tips	Protocol tips	Downstream tips
	Fot blot, dilute primary Ab at 1:1000 and for ICC or IF use 1:200

Protocol tips
Fot blot, dilute primary Ab at 1:1000 and for ICC or IF use 1:200

Manufacturer protocol Publication protocol 1 Relevant paper

Anti-LC3 pAb

MBL international corporation

Upstream tips	Protocol tips	Downstream tips
	Dilute at 1:2000 and incubate overnight at 4 °C

Protocol tips
Dilute at 1:2000 and incubate overnight at 4 °C

Manufacturer protocol Publication protocol 1 Relevant paper

LC3A/B (D3U4C) XP® Rabbit mAb #12741

Cell Signaling Technology

Upstream tips	Protocol tips	Downstream tips
	1:1000 dilution of primary antibody

Protocol tips
1:1000 dilution of primary antibody

Manufacturer protocol Publication protocol 1 Relevant paper

Phospho-mTOR (Ser2448) Antibody

Cell Signaling Technology

Upstream tips	Protocol tips	Downstream tips
	Dilute primary Ab at 1:1000

Protocol tips
Dilute primary Ab at 1:1000

Manufacturer protocol Publication protocol 1 Relevant paper

Phospho-eIF2α (Ser51) (D9G8) XP® Rabbit mAb #3398

Cell Signaling Technology

Upstream tips	Protocol tips	Downstream tips
	Dilute primary Ab at 1:1000

Protocol tips
Dilute primary Ab at 1:1000

Manufacturer protocol Publication protocol 1 Relevant paper

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