重组Anti-SQSTM1 / p62抗体[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.

ab109012 staining SQSTM1/p62 (autophagosome) in control HeLa cells treated with ethanol 0.1%  (left panels) and SQSTM1/p62 in HeLa cells treated with 1uM bafilomycin A1 (ab120497) for 18hrs (right panels). 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).

Lanes 1 - 4: Merged signal (red and green). Green - ab109012 observed at 55 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab109012 was shown to specifically react with SQSTM1 in wild-type HAP1 cells. No band was observed when SQSTM1 knockout samples were used. Wild-type and SQSTM1 knockout samples were subjected to SDS-PAGE, Ab109012 and ab8245 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1/10,000 dilution and 1/20,000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/20,000 dilution for 1 hour at room temperature before imaging.

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.

Purified ab109012 staining SQSTM1 in wild-type HAP1 cells (top panel) and SQSTM1 knockout HAP1 cells (bottom panel). The cells were fixed with 100% 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 1μg/ml and ab195889 at 1/250 dilution (shown in pseudocolour red) 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). Nuclear DNA was labelled in blue with DAPI.

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

Overlay histogram showing HeLa cells stained with unpurified ab109012 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab109012, 1/100 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluor^® 488 goat anti-rabbit IgG (H+L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.

Unpurified ab109012 staining SQSTM1/p62 in HeLa cells +/- Chloroquine (50μM, 24 hours). The cells were fixed with 100% methanol (5 min), 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 overnight at +4°C with ab109012 at 1μg/ml and ab195889, Mouse monoclonal to alpha Tubulin (Alexa Fluor^® 594), at 1/250 dilution (shown in pseudocolor red) 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). Nuclear DNA was labelled with DAPI (shown in blue).

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