SQSTM1 Antibody (D-3)

Protocol tips

Upstream tips	Protocol tips	Downstream tips
SDS-PAGE gels (8.5 or 12%) containing 25 µg of protein per well were transferred to 0.45 µm nitrocellulose (Bio-Rad 1620115) using constant voltage (100V) for 1 hr at 4°C in 1X transfer buffer with 10% ethanol. After transferring, the nitrocellulose membrane was blocked with 5% milk 1X TBST at room temperature for 30 minutes. The membranes were then washed three times, five minutes each, with 1X TBST.	Primary incubation with antibodies was done overnight at 4°C. After primary incubation, the blots were washed five times, five minutes each, with 1X TBST and then incubated in secondary antibody in 1% milk TBST for thirty minutes at room temperature. After secondary antibody incubation, the blots were washed five times, five minutes each, with 1X TBST and then once with 1X TBS for ten minutes.

Upstream tips
SDS-PAGE gels (8.5 or 12%) containing 25 µg of protein per well were transferred to 0.45 µm nitrocellulose (Bio-Rad 1620115) using constant voltage (100V) for 1 hr at 4°C in 1X transfer buffer with 10% ethanol. After transferring, the nitrocellulose membrane was blocked with 5% milk 1X TBST at room temperature for 30 minutes. The membranes were then washed three times, five minutes each, with 1X TBST.
Protocol tips
Primary incubation with antibodies was done overnight at 4°C. After primary incubation, the blots were washed five times, five minutes each, with 1X TBST and then incubated in secondary antibody in 1% milk TBST for thirty minutes at room temperature. After secondary antibody incubation, the blots were washed five times, five minutes each, with 1X TBST and then once with 1X TBS for ten minutes.

Publication protocol

SDS-PAGE gels (8.5 or 12%) containing 25 µg of protein per well were transferred to 0.45 µm nitrocellulose (Bio-Rad 1620115) using constant voltage (100V) for 1 hr at 4°C in 1X transfer buffer with 10% ethanol. After transferring, the nitrocellulose membrane was blocked with 5% milk 1X TBST at room temperature for 30 minutes. The membranes were then washed three times, five minutes each, with 1X TBST. Primary incubation with antibodies was done overnight at 4°C. After primary incubation, the blots were washed five times, five minutes each, with 1X TBST and then incubated in secondary antibody in 1% milk TBST for thirty minutes at room temperature. After secondary antibody incubation, the blots were washed five times, five minutes each, with 1X TBST and then
once with 1X TBS for ten minutes. TBS was removed, and the blots were incubated in ClarityTM Western ECL substrate (Bio-Rad 1705061) and imaged on the Bio-Rad ChemiDocTM Touch Imaging System using the chemiluminescence setting with 2x27 binning. Ladder images were taken using the colorimetric setting. After development, the membranes were washed with 1X TBST twice for five minutes each. 1:1000 β-actin HRP (Santa Cruz sc-47778 HRP) in 1% milk TBST was added and incubated for 1.5 hr at room
temperature. Developing occurred the same as after secondary antibody incubation.

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