Anti-SQSTM1 / p62 antibody (ab155686)

ab155686 staining SQSTM1/p62 (autophagosome) in control HeLa cells (left panel) and SQSTM1/p62 in HeLa cells treated with 1uM bafilomycin A1 (ab120497) for 18hrs (right panel). The cells were fixed with methanol (5min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab155686 at 1/500 dilution and ab7291 (Tubulin) at 1/1000 dilution overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 μg/ml (shown in green) and a goat secondary antibody to mouse IgG (Alexa Fluor® 594) (ab150120) at 2 μg/ml (shown in pseudo color red).  Nuclear DNA was labelled in blue with DAPI.

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

10% gel.

Running condition: 80V, 15min; 140V, 40min.

Transfer condition: Semi-dry, 18 V, 60min (Nitrocellulose membrane).

Blocking condition: 5% non-fat milk in TBST, RT, 60min.

Primary antibody incubation: 4°C overnight.

Secondary antibody incubation: Room temperature for 1 hour.

Washing condition: 5 ml TBST, 4 x 5min.

ECL exposure. 

Immunocytochemistry/Immunofluorescent analysis of HeLa cells mock (left) and treated with 50μM Chloroquine for 24 hr (right) labeling SQSTM1/p62 (green) with ab155686 at 1/1000 dilution. Cells were fixed with 4% paraformaldehyde at RT for 15 min. Red: Phalloidin, a F-actin marker.

Immunoprecipitation of SQSTM1/p62 protein from HeLa whole cell extracts using 5 μg of ab155686.
Western blot analysis was performed using ab155686.
Anti-Rabbit IgG was used as a secondary reagent.

Paraffin-embedded human ovarian carcinoma tissue stained for SQSTM1/p62 with ab155686 (1/500) in Immunohistochemical analysis. Antigen retrieval: EDTA based buffer (pH 8) for 15 minutes. 

10% SDS-PAGE

4% paraformaldehyde-fixed HeLa cells stained for SQSTM1/p62 (blue) using ab155686 (1/100) in Flow Cytometry. The cells were fixed at 4°C for 5 minutes. The brown line is unlabelled sample. Acquisition of >20,000 events were collected using Argon ion laser (488nm) and 525/30 bandpass filter.

Immunocytochemistry/ Immunofluorescence analysis of HepG2 cells treated with 3μM thapsigargin for 12 hours (right) and mock (left) labeling SQSTM1/p62 with ab155686 at 1:500 (green). Cells were fixed in ice-cold MeOH for 10 minutes, permeabilized with cooled acetone for 1 minute. Cells were co-stained with Hoechst (blue).

10% SDS-PAGE