Anti-SQSTM1 / p62 antibody [EPR4844] - BSA and Azide free

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-SQSTM1 / p62 antibody [EPR4844] - BSA and Azide free (AB219581)

Clone EPR4844 (ab219581) has been successfully conjugated by Abcam. This image was generated using Anti-SQSTM1 / p62 antibody [EPR4844] - Autophagosome Marker (Alexa Fluor® 488). Please refer to ab185015 for protocol details.

ab185015 staining SQSTM1 in wild-type HAP1 cells, untreated and chloroquine-treated (50μM, 24 hours) and chloroquine-treated SQSTM1 knockout HAP1 cells. The cells were fixed with 4% formaldehyde (10min), 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 ab185015 at 1/500 dilution (shown in green) and ab195889, Mouse monoclonal to alpha Tubulin (Alexa Fluor® 594), at 1/250 dilution (shown in pseudocolor red). Nuclear DNA was labelled with DAPI (shown in blue).

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

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-SQSTM1 / p62 antibody [EPR4844] - BSA and Azide free (AB219581)

This data was developed using the same antibody clone in a different buffer formulation (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.

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-SQSTM1 / p62 antibody [EPR4844] - BSA and Azide free (AB219581)

Intracellular Flow Cytometry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling SQSTM1 / p62 with purified ab109012 at 1/50 dilution (10μg/ml) (Red). Cells were fixed with 80% Methanol and permeabilised with 0.1% Tween-20. A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) secondary antibody was used at 1/2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab109012).

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-SQSTM1 / p62 antibody [EPR4844] - BSA and Azide free (AB219581)

Purified 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).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab109012).

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-SQSTM1 / p62 antibody [EPR4844] - BSA and Azide free (AB219581)

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).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab109012).

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-SQSTM1 / p62 antibody [EPR4844] - BSA and Azide free (AB219581)

Clone EPR4844 (ab219581) has been successfully conjugated by Abcam. This image was generated using Anti-SQSTM1 / p62 antibody [EPR4844] (Alexa Fluor® 647). Please refer to ab194721 for protocol details.

ab194721 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 ab194721 at 1/500 (shown in red) and ab195887 at 1/250 dilution (shown in green). Nuclear DNA was labelled in blue with DAPI.

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

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-SQSTM1 / p62 antibody [EPR4844] - BSA and Azide free (AB219581)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab109012).

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-SQSTM1 / p62 antibody [EPR4844] - BSA and Azide free (AB219581)

Clone EPR4844 (ab219581) has been successfully conjugated by Abcam. This image was generated using Anti-SQSTM1 / p62 antibody [EPR4844] - Autophagosome Marker (PE). Please refer to ab225094 for protocol details.

Overlay histogram showing HeLa cells untreated (red line) and HeLa cells treated with Chloroquine, 50μM, 24 hours, (green line) stained with ab225094. The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS / 10% normal goat serum to block non-specific protein-protein interactions followed by the antibody (ab225094, 1/10000 dilution) for 30 min at 22°C.

Isotype control antibody (black line) was Rabbit IgG (monoclonal) Phycoerythrin (ab209478) used at the same concentration and conditions as the primary antibody. Unlabelled sample (blue line) was also used as a control.

Acquisition of >5,000 events were collected using a 50 mW Yellow/Green laser (561nm) and 586/15 bandpass filter.

• WB

Collaborator

Western blot - Anti-SQSTM1 / p62 antibody [EPR4844] - BSA and Azide free (AB219581)

This data was developed using the same antibody in a different buffer formulation (ab109012).

ab109012 was shown to react with SQSTM1 in wild-type U-2 OS cells in Western blot with loss of signal observed in a SQSTM1 knockout cell line. Wild-type U-2 OS and SQSTM1 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 5% milk in TBST for 1 hr before incubation with ab109012 overnight at 4 °C at a 1/10000 dilution. Blots were incubated with goat anti-rabbit HRP secondary antibodies at 0.2ug/mL before imaging. These data were provided by YCharOS Inc., an open science company with the mission of characterizing commercially available antibody reagents for all human proteins. Abcam and YCharOS are working together to help address the reproducibility crisis by enabling the life science community to better evaluate commercially available antibodies.

All lanes:

Western blot - Anti-SQSTM1 / p62 antibody [EPR4844] - Autophagosome Marker (<a href='/en-us/products/primary-antibodies/sqstm1-p62-antibody-epr4844-autophagosome-marker-ab109012'>ab109012</a>) at 1/10000 dilution

Lane 1:

Wild-type U-2 OS cell lysate at 20 µg

Lane 2:

SQSTM1 knockout U-2 OS cell lysate at 20 µg

Predicted band size: 47 kDa

false

• WB

Lab

Western blot - Anti-SQSTM1 / p62 antibody [EPR4844] - BSA and Azide free (AB219581)

This data was developed using the same antibody clone in a different buffer formulation (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 (<a href='/en-us/products/primary-antibodies/sqstm1-p62-antibody-epr4844-autophagosome-marker-ab109012'>ab109012</a>) at 1/10000 dilution

Lane 1:

Wild-type HEK293T cell lysate at 20 µg

Lane 2:

SQSTM1 knockout HEK293T cell lysate at 20 µg

Lane 2:

Western blot - Human SQSTM1 (p62) knockout HEK-293T cell line (<a href='/en-us/products/cell-lines/human-sqstm1-p62-knockout-hek-293t-cell-line-ab255343'>ab255343</a>)

Predicted band size: 47 kDa

Observed band size: 64 kDa

false

• WB

Lab

Western blot - Anti-SQSTM1 / p62 antibody [EPR4844] - BSA and Azide free (AB219581)

This data was developed using ab109012, the same antibody clone in a different buffer formulation. Different batches of ab109012 were tested on MCF7 (Human breast adenocarcinoma epithelial cell) lysate at 0.4 µg/ml. 15 µg of lysate was loaded in each lane. Bands observed at 62 kDa.

All lanes:

Western blot - Anti-SQSTM1 / p62 antibody [EPR4844] - Autophagosome Marker (<a href='/en-us/products/primary-antibodies/sqstm1-p62-antibody-epr4844-autophagosome-marker-ab109012'>ab109012</a>)

Predicted band size: 47 kDa

false

• WB

Lab

Western blot - Anti-SQSTM1 / p62 antibody [EPR4844] - BSA and Azide free (AB219581)

Lanes 1 - 4 : Merged signal (red and green). Green - unpurifed 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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab109012).

All lanes:

Western blot - Anti-SQSTM1 / p62 antibody [EPR4844] - Autophagosome Marker (<a href='/en-us/products/primary-antibodies/sqstm1-p62-antibody-epr4844-autophagosome-marker-ab109012'>ab109012</a>) at 1/10000 dilution

Lane 1:

Wild-type HAP1 whole cell lysate at 20 µg

Lane 2:

SQSTM1 knockout HAP1 whole cell lysate at 20 µg

Lane 3:

HeLa whole cell lysate at 20 µg

Lane 4:

HepG2 whole cell lysate at 20 µg

Predicted band size: 47 kDa

false

• WB

Lab

Western blot - Anti-SQSTM1 / p62 antibody [EPR4844] - BSA and Azide free (AB219581)

This data was developed using ab109012, the same antibody clone in a different buffer formulation.

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 CRISPR/Cas9 edited cell line ab266871 (CRISPR/Cas9 edited 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 CRISPR/Cas9 edited samples were subjected to SDS-PAGE. ab109012 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were 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 (<a href='/en-us/products/primary-antibodies/sqstm1-p62-antibody-epr4844-autophagosome-marker-ab109012'>ab109012</a>)

Lane 1:

Wild-type HCT116 cell lysate at 20 µg

Lane 2:

SQSTM1 CRISPR/Cas9 edited 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 (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/10000 dilution

Predicted band size: 47 kDa

Observed band size: 55 kDa

false