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

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
SQSTM1 Antibody (D-3)

Santa Cruz Biotechnology

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.
JNK1 Antibody (F-3)

Santa Cruz Biotechnology

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)
LAMP1 (D2D11) XP® Rabbit mAb

Cell Signaling Technology

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
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.
LC3B Antibody

Novus Biologicals

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.
Beclin-1 (D40C5) Rabbit mAb

Cell Signaling Technology

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
LC3B (D11) XP® Rabbit mAb

Cell Signaling Technology

Upstream tips
Lyse cells in RIPA buffer with protein inhibitor
Protocol tips
Dilute primary Ab at 1:1000 and incubate overnight at 4°C.
Protocol tips
Incubate the membrane with diluted primary antibody in
blocking buffer and incubate overnight at 4C
eIF2α Antibody

Cell Signaling Technology

Protocol tips
Dilute primary Ab at 1:1000 and incubate with primary antibodies overnight at 4°C
Phospho-eIF2α (Ser51) Antibody

Cell Signaling Technology

Protocol tips
Dilute primary Ab at 1:1000 and incubate with primary antibodies overnight at 4°C
Atg7 (D12B11) Rabbit mAb

Cell Signaling Technology

Protocol tips
Dilute primary Ab at 1:1000 and incubate with gentle agitation overnight at 4°C
Upstream tips
Discarded working dilution samples if not used within
12 hours.
LC3B Rabbit Antibody

Cell Signaling Technology

Protocol tips
Dilute primary Ab at 1:1000 and incubate overnight at 4°C.
Anti-Beclin 1 (Human) pAb

MBL international corporation

Protocol tips
Dilute Primary Ab at 1∶2000 and incubate overnight at 4C
Atg7 Antibody

Cell Signaling Technology

Protocol tips
Dilute primary Ab at 1:1000 and incubate overnight at 4°C.
Phospho-mTOR (Ser2448) Antibody

Cell Signaling Technology

Protocol tips
Dilute primary Ab at 1:1000 and incubate overnight at 4°C.
mTOR (7C10) Rabbit mAb

Cell Signaling Technology

Protocol tips
Dilute primary Ab at 1:1000 and incubate overnight at 4°C
LAMP1 (C54H11) Rabbit mAb

Cell Signaling Technology

Protocol tips
Dilute primary Ab at 1:1000 and incubate overnight at 4°C
H4A3

Developmental Studies Hybridoma Bank

Protocol tips
For WB use concentration of 0.2-0.5 µg/ml for primary Ab
PI 3-kinase p85α Antibody (B-9)

Santa Cruz Biotechnology

Protocol tips
Dilute primary Ab at 1:200 and incubate at 4°C overnight
Atg3 Antibody

Cell Signaling Technology

Protocol tips
Dilute primary Ab at 1:1000 and incubate overnight at 4°C
Anti-p62 (SQSTM1) (Human) pAb

MBL international corporation

Protocol tips
Dilute Primary Ab at 1∶2000 and incubate for 1hr at RT
LC3A (D50G8) XP® Rabbit mAb

Cell Signaling Technology

Protocol tips
Dilute primary Ab at 1:1000
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