Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: SQSTM1/p62 knockout HAP1 cell lysate (20 µg)
Lane 3: HeLa cell lysate (20 µg)
Lane 4: HepG2 cell lysate (20 µg)
Lanes 1 - 4: Merged signal (red and green).

Green - target observed at 55 kDa. Red - loading control, ab8245, observed at 37 kDa.

This western blot image is a comparison between ab207305 and a competitor's discontinued goat polyclonal antibody.

 

 

All lanes : Anti-SQSTM1 / p62 antibody [EPR18351] (ab207305)

Lane 1 : Wild-type HCT116 cell lysate
Lane 2 : SQSTM1 knockout HCT116 cell lysate
Lane 3 : HepG2 cell lysate
Lane 4 : HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) at 1/10000 dilution

Predicted band size: 47 kDa
Observed band size: 55 kDa why is the actual band size different from the predicted?

Lanes 1-4: Merged signal (red and green). Green - ab207305 observed at 55 kDa. Red - loading control ab8245 observed at 36 kDa.

 ab207305 Anti-SQSTM1 / p62 antibody [EPR18351] 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. ab207305 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.

ab207305 staining SQSTM1 in wild-type HAP1 cells and knockout cells, untreated and chloroquine-treated (50μM, 24 hours). 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 with ab207305 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).

Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: SQSTM1/p62 knockout HAP1 cell lysate (20 µg)
Lane 3: HeLa cell lysate (20 µg)
Lane 4: HepG2 cell lysate (20 µg)
Lanes 1 - 4: Merged signal (red and green). Green - ab207305 observed at 55 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab207305 was shown to specifically react with SQSTM1/p62 in wild-type HAP1 cells. No band was observed when SQSTM1/p62 knockout samples were used. Wild-type and SQSTM1/p62 knockout samples were subjected to SDS-PAGE, ab207305 and ab8245 (loading control to GAPDH) were diluted 1/1000 and 1/10,000 respectively and incubated overnight at 4°C. 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 1/10,000 dilution for 1 hour at room temperature before imaging.

Immunohistochemical analysis of paraffin-embedded human hepatocellular carcinoma tissue labeling SQSTM1 / p62 with ab207305 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Cytoplasm staining on human hepatocellular carcinoma is observed. Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

ab207305 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 ab207305 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).

All lanes : Anti-SQSTM1 / p62 antibody [EPR18351] (ab207305) at 1/1000 dilution

Lane 1 : Human liver lysate
Lane 2 : Human kidney lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG Peroxidase Conjugate, specific to the non-reduced form of IgG at 1/10000 dilution

Predicted band size: 47 kDa
Observed band size: 62 kDa why is the actual band size different from the predicted?


Exposure time: 30 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

The MW observed is consistent with what has been described in the literature (PMID: PMC4344198).

All lanes : Anti-SQSTM1 / p62 antibody [EPR18351] (ab207305) at 1/5000 dilution

Lane 1 : HepG2 (Human liver hepatocellular carcinoma) whole cell lysate
Lane 2 : HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 47 kDa
Observed band size: 62 kDa why is the actual band size different from the predicted?


Exposure time: 30 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

The MW observed is consistent with what has been described in the literature (PMID: PMC4344198).

Immunohistochemical analysis of paraffin-embedded human lung cancer tissue labeling SQSTM1 / p62 with ab207305 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Cytoplasm and weakly nucleus staining on human lung cancer is observed. Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded human liver tissue labeling SQSTM1 / p62 with ab207305 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Negative staining on Hhman normal liver tissue. Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Flow cytometric analysis of 4% paraformaldehyde-fixed PC-3 (Human prostate adenocarcinoma cell line) cells labeling SQSTM1 / p62 with ab207305 at 1/100 dilution (red) compared with a rabbit monoclonal IgG isotype control (ab172730; black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody; blue). Goat Anti-Rabbit IgG (FITC) at 1/500 dilution was used as the secondary antibody.

SQSTM1 / p62 was immunoprecipitated from 1mg of HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysate with ab207305 at 1/40 dilution.
Western blot was performed from the immunoprecipitate using ab207305 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.
Lane 1: HeLa whole cell lysate 10ug (Input).
Lane 2: ab207305 IP in HeLa whole cell lysate.
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab207305 in HeLa whole cell lysate.
Blocking and dilution buffer and concentration: 5% NFDM/TBST.
Exposure time: 1 second.

SQSTM1 / p62 was immunoprecipitated from 1mg of HepG2 (Human liver hepatocellular carcinoma) whole cell lysate with ab207305 at 1/40 dilution.
Western blot was performed from the immunoprecipitate using ab207305 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.
Lane 1: HepG2 whole cell lysate 10ug (Input).
Lane 2: ab207305 IP in HepG2 whole cell lysate.
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab207305 in HepG2 whole cell lysate.
Blocking and dilution buffer and concentration: 5% NFDM/TBST.
Exposure time: 1 second.