Anti-SQSTM1 / p62 (phospho S349) antibody [EPR20451]

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-SQSTM1 / p62 (phospho S349) antibody [EPR20451] (AB211324)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cell line from cervix adenocarcinoma) cells, treated with 2μM MG-132 (ab141003) for 18 hours or untreated, labeling SQSTM1 / p62 (phospho S349) with ab211324 at 1/100 dilution, followed by Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) secondary antibody at 1/200 dilution (green).

Confocal image showing cytoplasmic staining on HeLa cell line. The expression increased after treatment with 2μM MG-132 (ab141003) for 18 hours.

The nuclear counterstain is DAPI (blue).

Tubulin is detected with ab195889 (Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594)) at 1/200 dilution (red).

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) at 1/1000 dilution.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-SQSTM1 / p62 (phospho S349) antibody [EPR20451] (AB211324)

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed HeLa (Human epithelial cell line from cervix adenocarcinoma) cells, treated with 2μM MG-132 (ab141003) for 18 hours (red) or untreated (green), labeling SQSTM1 / p62 (phospho S349) with ab211324 at 1/500 dilution compared with a rabbit monoclonal IgG isotype control (ab172730; black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody; blue). Goat anti rabbit IgG (Alexa Fluor^® 488) at 1/2000 dilution was used as the secondary antibody.

• IP

Lab

Immunoprecipitation - Anti-SQSTM1 / p62 (phospho S349) antibody [EPR20451] (AB211324)

SQSTM1 / p62 (phospho S349) was immunoprecipitated from 0.35 mg of HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate treated with 2μM MG-132 (ab141003) for 18h with ab211324 at 1/30 dilution.

Western blot was performed from the immunoprecipitate using ab211324 at 1/1000 dilution.

VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10,000 dilution.

Lane 1 : HeLa treated with 2μM MG-132 (ab141003) for 18h whole cell lysate, 10 μg (Input).
Lane 2 : ab211324 IP in HeLa treated with 2μM MG-132 (ab141003) for 18h whole cell lysate.
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab211324 in HeLa treated with 2μM MG-132 (ab141003) for 18h whole cell lysate.

Blocking and dilution buffer : 5% NFDM/TBST.

All lanes:

Immunoprecipitation - Anti-SQSTM1 / p62 (phospho S349) antibody [EPR20451] (ab211324)

Predicted band size: 47 kDa

false

Exposure time: 10s

• WB

Collaborator

Western blot - Anti-SQSTM1 / p62 (phospho S349) antibody [EPR20451] (AB211324)

ab211324 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 ab211324 overnight at 4 °C at a 1/1000 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 (phospho S349) antibody [EPR20451] (ab211324) at 1/1000 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

Unknown

Western blot - Anti-SQSTM1 / p62 (phospho S349) antibody [EPR20451] (AB211324)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-SQSTM1 / p62 (phospho S349) antibody [EPR20451] (ab211324) at 1/1000 dilution

Lane 1:

Untreated HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 10 µg

Lane 2:

HeLa whole cell lysate treated with 2μM MG-132 (<a href='/en-us/products/biochemicals/mg-132-proteasome-inhibitor-ab141003'>ab141003</a>) for 18 hours at 10 µg

Lane 3:

HeLa whole cell lysate treated with 2μM MG-132 (<a href='/en-us/products/biochemicals/mg-132-proteasome-inhibitor-ab141003'>ab141003</a>) for 18 hours, then treated with Alkaline Phosphatase for 1 hour at 10 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 1/100000 dilution

Predicted band size: 47 kDa

Observed band size: 62 kDa

false

Exposure time: 1min

• WB

Lab

Western blot - Anti-SQSTM1 / p62 (phospho S349) antibody [EPR20451] (AB211324)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-SQSTM1 / p62 (phospho S349) antibody [EPR20451] (ab211324) at 1/1000 dilution

Lane 1:

Untreated C6 (Rat glial tumor cell line) whole cell lysate at 10 µg

Lane 2:

C6 whole cell lysate treated with 2μM MG-132 (<a href='/en-us/products/biochemicals/mg-132-proteasome-inhibitor-ab141003'>ab141003</a>) for 18 hours at 10 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 1/100000 dilution

Predicted band size: 34 kDa,47 kDa,76 kDa

Observed band size: 42 kDa,62 kDa,76 kDa

false

Exposure time: 1min

• WB

Lab

Western blot - Anti-SQSTM1 / p62 (phospho S349) antibody [EPR20451] (AB211324)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-SQSTM1 / p62 (phospho S349) antibody [EPR20451] (ab211324) at 1/1000 dilution

Lane 1:

Untreated NIH/3T3 (Mouse embryonic fibroblast cell line) whole cell lysate at 10 µg

Lane 2:

NIH/3T3 whole cell lysate treated with 2μM MG-132 (<a href='/en-us/products/biochemicals/mg-132-proteasome-inhibitor-ab141003'>ab141003</a>) for 18 hours at 10 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 1/100000 dilution

Predicted band size: 47 kDa

Observed band size: 62 kDa

false

Exposure time: 4s