SQSTM1 Antibody (D-3)

Cell cytotoxicity / Proliferation assay cell type - K562

Experiment
Cell cytotoxicity / Proliferation assay cell type - K562
Product
SQSTM1 Antibody (D-3) from Santa Cruz Biotechnology
Manufacturer
Santa Cruz Biotechnology

Protocol tips

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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Manufacturer protocol

Download the product protocol from Santa Cruz Biotechnology for SQSTM1 Antibody (D-3) below.

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