Immunoprecipitation - Anti-SQSTM1 / p62 antibody [EPR4844] - Autophagosome Marker (ab109012)
Immunoprecipitation of SQSTM1 in U2OSn cells. Lysates were prepared and immunoprecipitation was performed using 1.0 μg of ab109012 pre-coupled to prot.A-Sepharose beads. Samples were washed and processed for western blot with GTX629890 at 1/5000. This data was kindly provided by the YCharOS Inc., an open science company with the mission of characterizing every commercially available antibody reagent. Abcam are working with YCharOS to support their mission of antibody characterisation using knock out cell lines.
All lanes: Immunoprecipitation - Anti-SQSTM1 / p62 antibody [EPR4844] - Autophagosome Marker (ab109012)
Predicted band size: 47 kDa
Immunocytochemistry/ Immunofluorescence - Anti-SQSTM1 / p62 antibody [EPR4844] - Autophagosome Marker (ab109012)
ab109012 staining SQSTM1 in wild-type Hap1 cells and SQSTM1 knockout Hap1 cells treated with chloroquine (ab142116, 50μM for 24 hrs). The cells were fixed with 100% methanol (5 min) or with 4% paraformaldehyde (10 min) then 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 ab109012 at 0.1μg/ml concentration and ab7291 (Mouse monoclonal to alpha 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 red). Nuclear DNA was labelled in blue with DAPI.
Image was taken with a confocal microscope (Leica-Microsystems TCS SP8).
Residual signal is observed in KO cells when paraformaldehyde is used for fixation, therefore we recommend using methanol fixation with this antibody. Alternatively, please use ab240635 or ab207305 which have been KO validated in both paraformaldehyde and methanol fixed cells.
Western blot - Anti-SQSTM1 / p62 antibody [EPR4844] - Autophagosome Marker (ab109012)
Lanes 1-4: Merged signal (red and green). Green - ab109012 observed at 55 kDa. Red - loading control ab8245 observed at 36 kDa.
ab109012 Anti-SQSTM1 / p62 antibody [EPR4844] - Autophagosome Marker was shown to specifically react with SQSTM1 / p62 in wild-type HCT116 cells. The band observed in knockout cell line ab266871 (knockout cell lysate ab257052) lane below 55 kDa may represent truncated forms and cleaved fragments. This has not been investigated further. Wild-type and SQSTM1 / p62 knockout samples were subjected to SDS-PAGE. ab109012 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.
All lanes: Western blot - Anti-SQSTM1 / p62 antibody [EPR4844] - Autophagosome Marker (ab109012)
Lane 1: Wild-type HCT116 cell lysate at 20 µg
Lane 2: SQSTM1 knockout HCT116 cell lysate at 20 µg
Lane 3: HepG2 cell lysate at 20 µg
Lane 4: HeLa cell lysate at 20 µg
Secondary
All lanes: Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) at 1/10000 dilution
Predicted band size: 47 kDa
Observed band size: 55 kDa
Immunocytochemistry/ Immunofluorescence - Anti-SQSTM1 / p62 antibody [EPR4844] - Autophagosome Marker (ab109012)
ab109012 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 ab109012 at 5ug/ml 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).
Western blot - Anti-SQSTM1 / p62 antibody [EPR4844] - Autophagosome Marker (ab109012)
Lanes 1- 2: Merged signal (red and green). Green - ab109012 observed at 64 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.
ab109012 was shown to react with SQSTM1/p62 in wild-type HEK293T cells in western blot. Loss of signal was observed when knockout cell line ab255343 (knockout cell lysate ab263770) was used. Wild-type HEK293T and SQSTM1 knockout HEK293T cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab109012 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 10000 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye®800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye®680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.
All lanes: Western blot - Anti-SQSTM1 / p62 antibody [EPR4844] - Autophagosome Marker (ab109012) at 1/10000 dilution
Lane 1: Wild-type HEK293T cell lysate at 20 µg
Lane 2: SQSTM1 knockout HEK293T cell lysate at 20 µg
Performed under reducing conditions.
Predicted band size: 47 kDa
Observed band size: 64 kDa
