Anti-SQSTM1 / p62 at D329 (N-terminal) antibody [EPR29289-46]

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-SQSTM1 / p62 at D329 (N-terminal) antibody [EPR29289-46] (AB325681)

Immunofluorescent analysis of 100% methanol-fixed, 0.1% TritonX-100 permeabilized HeLa(human cervical adenocarcinoma epithelial cell) untreated, HeLa treated with TNF alpha (100 ng/ml) and IKK-16(1uM) for 4 hours and HeLa treated with TNF alpha (100 ng/ml), IKK-16(1uM) and Z-VAD-FM(20uM) for 4 hours cells labelling SQSTM1 / p62 with ab325681 at 1/50 (10.24 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (Green).

Confocal image showing increased cytoplasmic staining in HeLa cells treated with TNF alpha (100 ng/ml) and IKK-16 (1uM) for 4 hours (panel B). No staining was observed in untreated HeLa cells (panel A), or HeLa cells treated with TNF alpha (100 ng/ml), IKK-16 (1uM) and Z-VAD-FM (20uM) for 4 hours (panel C). The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue). Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

ab7291 Anti-alpha Tubulin mouse monoclonal antibody was used to counterstain tubulin at 1/1000 dilution, followed by ab150120 Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) at 1/1000 dilution (Magenta).

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-SQSTM1 / p62 at D329 (N-terminal) antibody [EPR29289-46] (AB325681)

Immunofluorescent analysis of 100% methanol-fixed, 0.1% TritonX-100 permeabilized wildtype HAP1 (human chronic myelogenous leukemia near-haploid cell) untreated, wildtype HAP1 treated with TNF alpha (100 ng/ml) and IKK-16 (1µM) for 4 hours, SQSTM1 knockout HAP1 untreated and SQSTM1 knockout HAP1 treated with TNF alpha (100 ng/ml) and IKK-16 (1µM) for 4 hours labelling SQSTM1 / p62 with ab325681 at 1/50 (10.24 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (Green).

Confocal image showing increased cytoplasmic staining in wildtype HAP1 cells treated with TNF alpha (100 ng/ml) and IKK-16 (1uM) for 4 hours (panel B). No staining was observed in untreated wildtype HAP1 cells (panel A), untreated SQSTM1 knockout HAP1 cells (panel C), or SQSTM1 knockout HAP1 cells treated with TNF alpha (100 ng/ml) and IKK-16 (1uM) for 4 hours (panel D). The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue). Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

ab7291 Anti-alpha Tubulin mouse monoclonal antibody was used to counterstain tubulin at 1/1000 dilution, followed by ab150120 Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) at a 1/1000 dilution (Magenta).

• WB

Lab

Western blot - Anti-SQSTM1 / p62 at D329 (N-terminal) antibody [EPR29289-46] (AB325681)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

Total protein Anti-SQSTM1 antibody [EPR18351] (ab207305) (1 : 1000).

All lanes:

Western blot - Anti-SQSTM1 / p62 at D329 (N-terminal) antibody [EPR29289-46] (ab325681) at 1/1000 dilution

Lane 1:

Untreated HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

HeLa treated with 10ng/ml TNF alpha and 1uM IKK-16 for 4 hours whole cell lysate at 20 µg

Lane 3:

HeLa treated with 10ng/ml TNF alpha, 1uM IKK-16 and 20uM Z-VAD-FMK for 4 hours whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 40 kDa,36 kDa

false

Exposure time: 10s

• WB

Lab

Western blot - Anti-SQSTM1 / p62 at D329 (N-terminal) antibody [EPR29289-46] (AB325681)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

In Western blot, ab325681 was shown to bind specifically to SQSTM1. Target of interest was observed at 40 kDa in TNF alpha and IKK-16 treated wild-type HAP1 cell lysates (lane 2) with no signal observed at this size in the SQSTM1 knockout cell line (lanes 3-4, knockout cell line ab240635).

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

Total protein Anti-SQSTM1 antibody [EPR18351] (ab207305) (1 : 1000).

All lanes:

Western blot - Anti-SQSTM1 / p62 at D329 (N-terminal) antibody [EPR29289-46] (ab325681) at 1/1000 dilution

Lane 1:

Untreated parental HAP1 (human chronic myelogenous leukemia near-haploid cell) whole cell lysate at 60 µg

Lane 2:

HAP1 treated with 10ng/ml TNF alpha and 1uM IKK-16 for 4 hours whole cell lysate at 60 µg

Lane 3:

Untreated SQSTM1 knockout HAP1 whole cell lysate at 60 µg

Lane 4:

SQSTM1 knockout HAP1 treated with 10ng/ml TNF alpha and 1uM IKK-16 for 4 hours whole cell lysate at 60 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 40 kDa,36 kDa

false

Exposure time: 180s

• I-ELISA

Lab

Indirect ELISA - Anti-SQSTM1 / p62 at D329 (N-terminal) antibody [EPR29289-46] (AB325681)

Indirect ELISA analysis of ab325681 at 1000-0 ng/ml. The Secondary antibody used was Alkaline Phosphatase-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L) at 1 : 2500 dilution dilution.

Antigen : cleaved human SQSTM1 at D329 (N-terminal);Full length human SQSTM1.

Antigen concentration : 1000 ng/ml

This antibody does not cross-react with full length human SQSTM1.