Autophagy assay cell type - SH-SY5Y

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 24 matching solutions for this experiment

Purified Mouse Anti-p62 Ick ligand Clone 3/P62 LCK LIGAND (RUO)

BD Biosciences

Upstream tips	Protocol tips	Downstream tips
For WB, lyse cells in on ice in 1% (v/v) Triton X‐100 in PBS supplemented with protease and phosphatase inhibitors or 0.1% (v/v) sodium dodecyl sulphate (SDS), 10 mM Tris pH 7.4, 150 mM NaCl supplemented with DNase and protease/phosphatase inhibitors	Incubate primary Ab overnight at 4°C

Upstream tips
For WB, lyse cells in on ice in 1% (v/v) Triton X‐100 in PBS supplemented with protease and phosphatase inhibitors or 0.1% (v/v) sodium dodecyl sulphate (SDS), 10 mM Tris pH 7.4, 150 mM NaCl supplemented with DNase and protease/phosphatase inhibitors
Protocol tips
Incubate primary Ab overnight at 4°C

Manufacturer protocol Publication protocol 1 Relevant paper

SQSTM1 Antibody (D-3)

Santa Cruz Biotechnology

Upstream tips	Protocol tips	Downstream tips
Lyse cells in a buffer solution containing 50mM Tris/HCl, pH7.8, 150 mM NaCl, 1% Triton X-100, 100 uM PMSF, a 1X dilution of complete protease and a phosphotase inhibitor mixture	Dilute primary Ab at 1:1000 and incubate overnight at 4 °C.

Upstream tips
Lyse cells in a buffer solution containing 50mM Tris/HCl, pH7.8, 150 mM NaCl, 1% Triton X-100, 100 uM PMSF, a 1X dilution of complete protease and a phosphotase inhibitor mixture
Protocol tips
Dilute primary Ab at 1:1000 and incubate overnight at 4 °C.

Manufacturer protocol Publication protocol 1 Relevant paper

JNK1 Antibody (F-3)

Santa Cruz Biotechnology

Upstream tips	Protocol tips	Downstream tips
Lyse cells in lysis buffer (100 mm NaCl, 50 mm Tris-HCl, 1 mm EGTA, 10 mmMgCl2, pH 7.2) containing 1 % Triton X-100, phosphatase, and protease inhibitor mixture	Dilute primary Ab for western blotting (starting dilution 1:200, dilution range 1:100-1:1000)

Upstream tips
Lyse cells in lysis buffer (100 mm NaCl, 50 mm Tris-HCl, 1 mm EGTA, 10 mmMgCl2, pH 7.2) containing 1 % Triton X-100, phosphatase, and protease inhibitor mixture
Protocol tips
Dilute primary Ab for western blotting (starting dilution 1:200, dilution range 1:100-1:1000)

Manufacturer protocol Publication protocol 1 Relevant paper

LAMP1 (D2D11) XP® Rabbit mAb

Cell Signaling Technology

Upstream tips	Protocol tips	Downstream tips
Lyse cells in a buffer solution containing 50mM Tris/HCl, pH7.8, 150 mM NaCl, 1% Triton X-100, 100 uM PMSF, a 1X dilution of complete protease and a phosphotase inhibitor mixture	Dilute primary Ab at 1:1000 and incubate overnight at 4°C

Upstream tips
Lyse cells in a buffer solution containing 50mM Tris/HCl, pH7.8, 150 mM NaCl, 1% Triton X-100, 100 uM PMSF, a 1X dilution of complete protease and a phosphotase inhibitor mixture
Protocol tips
Dilute primary Ab at 1:1000 and incubate overnight at 4°C

Manufacturer protocol Publication protocol 1 Relevant paper

Anti-LC3B antibody produced in rabbit

Sigma-Aldrich

Upstream tips	Protocol tips	Downstream tips
	Protein lysates were separated by SDS-PAGE and probed with the following antibodies	Relative levels of protein to ACTB loading control were quantified by densitometry using ImageJ, and normalized to the lane with the highest signal.

Protocol tips
Protein lysates were separated by SDS-PAGE and probed with the following antibodies
Downstream tips
Relative levels of protein to ACTB loading control were quantified by densitometry using ImageJ, and normalized to the lane with the highest signal.

Manufacturer protocol Publication protocol 1 Relevant paper

LC3B Antibody

Novus Biologicals

Upstream tips	Protocol tips	Downstream tips
	.Dilute the rabbit anti-LC3B primary antibody (~2 ug/mL) in blocking buffer and incubate the membrane for 1 hour at room temperature.

Protocol tips
.Dilute the rabbit anti-LC3B primary antibody (~2 ug/mL) in blocking buffer and incubate the membrane for 1 hour at room temperature.

Manufacturer protocol Publication protocol 1 Relevant paper

Beclin-1 (D40C5) Rabbit mAb

Cell Signaling Technology

Upstream tips	Protocol tips	Downstream tips
Lyse cells in RIPA buffer with protein inhibitor	Dilute primary Ab at 1:1000 and incubate overnight at 4°C with gentle shaking

Upstream tips
Lyse cells in RIPA buffer with protein inhibitor
Protocol tips
Dilute primary Ab at 1:1000 and incubate overnight at 4°C with gentle shaking

Manufacturer protocol Publication protocol 1 Relevant paper

LC3B (D11) XP® Rabbit mAb

Cell Signaling Technology

Upstream tips	Protocol tips	Downstream tips
Lyse cells in RIPA buffer with protein inhibitor	Dilute primary Ab at 1:1000 and incubate overnight at 4°C.

Upstream tips
Lyse cells in RIPA buffer with protein inhibitor
Protocol tips
Dilute primary Ab at 1:1000 and incubate overnight at 4°C.

Manufacturer protocol Publication protocol 1 Relevant paper

Anti-Apg7 antibody [EPR6251]

Abcam

Upstream tips	Protocol tips	Downstream tips
	Incubate the membrane with diluted primary antibody in blocking buffer and incubate overnight at 4C

Protocol tips
Incubate the membrane with diluted primary antibody in blocking buffer and incubate overnight at 4C

Manufacturer protocol Publication protocol 1 Relevant paper

eIF2α Antibody

Cell Signaling Technology

Upstream tips	Protocol tips	Downstream tips
	Dilute primary Ab at 1:1000 and incubate with primary antibodies overnight at 4°C

Protocol tips
Dilute primary Ab at 1:1000 and incubate with primary antibodies overnight at 4°C

Manufacturer protocol Publication protocol 1 Relevant paper

Phospho-eIF2α (Ser51) Antibody

Cell Signaling Technology

Upstream tips	Protocol tips	Downstream tips
	Dilute primary Ab at 1:1000 and incubate with primary antibodies overnight at 4°C

Protocol tips
Dilute primary Ab at 1:1000 and incubate with primary antibodies overnight at 4°C

Manufacturer protocol Publication protocol 1 Relevant paper

Atg7 (D12B11) Rabbit mAb

Cell Signaling Technology

Upstream tips	Protocol tips	Downstream tips
	Dilute primary Ab at 1:1000 and incubate with gentle agitation overnight at 4°C

Protocol tips
Dilute primary Ab at 1:1000 and incubate with gentle agitation overnight at 4°C

Manufacturer protocol Publication protocol 1 Relevant paper

Anti-LC3 antibody produced in rabbit

Sigma-Aldrich

Upstream tips	Protocol tips	Downstream tips
Discarded working dilution samples if not used within 12 hours.

Upstream tips
Discarded working dilution samples if not used within 12 hours.

Manufacturer protocol Publication protocol 1 Relevant paper

LC3B Rabbit Antibody

Cell Signaling Technology

Upstream tips	Protocol tips	Downstream tips
	Dilute primary Ab at 1:1000 and incubate overnight at 4°C.

Protocol tips
Dilute primary Ab at 1:1000 and incubate overnight at 4°C.

Manufacturer protocol Publication protocol 1 Relevant paper

Anti-Beclin 1 (Human) pAb

MBL international corporation

Upstream tips	Protocol tips	Downstream tips
	Dilute Primary Ab at 1∶2000 and incubate overnight at 4C

Protocol tips
Dilute Primary Ab at 1∶2000 and incubate overnight at 4C

Manufacturer protocol Publication protocol 1 Relevant paper

Atg7 Antibody

Cell Signaling Technology

Upstream tips	Protocol tips	Downstream tips
	Dilute primary Ab at 1:1000 and incubate overnight at 4°C.

Protocol tips
Dilute primary Ab at 1:1000 and incubate overnight at 4°C.

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 and incubate overnight at 4°C.

Protocol tips
Dilute primary Ab at 1:1000 and incubate overnight at 4°C.

Manufacturer protocol Publication protocol 1 Relevant paper

mTOR (7C10) Rabbit mAb

Cell Signaling Technology

Upstream tips	Protocol tips	Downstream tips
	Dilute primary Ab at 1:1000 and incubate overnight at 4°C

Protocol tips
Dilute primary Ab at 1:1000 and incubate overnight at 4°C

Manufacturer protocol Publication protocol 1 Relevant paper

LAMP1 (C54H11) Rabbit mAb

Cell Signaling Technology

Upstream tips	Protocol tips	Downstream tips
	Dilute primary Ab at 1:1000 and incubate overnight at 4°C

Protocol tips
Dilute primary Ab at 1:1000 and incubate overnight at 4°C

Manufacturer protocol Publication protocol 1 Relevant paper

H4A3

Developmental Studies Hybridoma Bank

Upstream tips	Protocol tips	Downstream tips
	For WB use concentration of 0.2-0.5 µg/ml for primary Ab

Protocol tips
For WB use concentration of 0.2-0.5 µg/ml for primary Ab

Manufacturer protocol Publication protocol 1 Relevant paper

PI 3-kinase p85α Antibody (B-9)

Santa Cruz Biotechnology

Upstream tips	Protocol tips	Downstream tips
	Dilute primary Ab at 1:200 and incubate at 4°C overnight

Protocol tips
Dilute primary Ab at 1:200 and incubate at 4°C overnight

Manufacturer protocol Publication protocol 1 Relevant paper

Atg3 Antibody

Cell Signaling Technology

Upstream tips	Protocol tips	Downstream tips
	Dilute primary Ab at 1:1000 and incubate overnight at 4°C

Protocol tips
Dilute primary Ab at 1:1000 and incubate overnight at 4°C

Manufacturer protocol Publication protocol 1 Relevant paper

Anti-p62 (SQSTM1) (Human) pAb

MBL international corporation

Upstream tips	Protocol tips	Downstream tips
	Dilute Primary Ab at 1∶2000 and incubate for 1hr at RT

Protocol tips
Dilute Primary Ab at 1∶2000 and incubate for 1hr at RT

Manufacturer protocol Publication protocol 1 Relevant paper

LC3A (D50G8) XP® Rabbit mAb

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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