All lanes : Anti-SQSTM1 / p62 antibody [3/P62 LCK LIGAND] (ab280086) at 1/1000 dilution

Lane 1 : Wild-type HAP1 (human chronic myelogenous leukemia near-haploid cell line), whole cell lysate
Lane 2 : SQSTM1 knockout HAP1 (human chronic myelogenous leukemia near-haploid cell line), whole cell lysate
Lane 3 : HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate
Lane 4 : MCF7 (human breast adenocarcinoma epithelial cell), whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Mouse IgG H&L (IRDye® 800CW) (ab216772) and Goat Anti-Rabbit IgG H&L (IRDye® 680RD) (ab216777) at 1/10000 dilution

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

Blocking and diluting buffer and concentration: Intercept® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS

Lanes 1 - 4: Merged signal (red and green). Green - ab280086 observed at 62 kDa. Red - loading control ab181602 (Rabbit monoclonal [EPR16891] to GAPDH) observed at 36 kDa.

Lanes 1-2: ab280086 Anti-SQSTM1/p62 antibody was shown to react with SQSTM1 in HAP1 cells in Western blot. Loss of signal was observed when SQSTM1 knockout sample was used. Wild-type and SQSTM1 knockout samples were subjected to SDS-PAGE. ab280086 and Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) were incubated at 4°C overnight at 1/1000 dilution and 1/20000 dilution respectively.

Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 680CW) preadsorbed (ab216777) and Goat anti-Mouse IgG H&L (IRDye® 800RD) preadsorbed (ab216772) secondary antibodies at 1 in 10000 dilution for 1 hour at room temperature before imaging. 

All lanes : Anti-SQSTM1 / p62 antibody [3/P62 LCK LIGAND] (ab280086) at 1/1000 dilution

Lane 1 : HeLa (human cervix adenocarcinoma epithelial cell ), whole cell lysate
Lane 2 : HEK-293 (human embryonic kidney epithelial cell), whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Peroxidase-Conjugated Goat anti-Mouse IgG (H+L) at 1/10000 dilution

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

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

The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID: 24086455).

Lysates were made freshly and used in WB immediately to minimize protein degradation.

Exposure time: 15 seconds

Immunohistochemical analysis of paraffin-embedded Human stomach carcinoma tissue labeling SQSTM1 / p62 with ab280086 at 1/1000 dilution followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on human stomach carcinoma. The section was incubated with ab280086 for 30 mins at room temperature and followed by mouse specific IgG antibody (ab125913) for 8mins. The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used.

Heat mediated antigen retrieval with Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins

Immunohistochemical analysis of paraffin-embedded Human lung carcinoma tissue labeling SQSTM1 / p62 with ab280086 at 1/1000 dilution followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on human lung carcinoma. The section was incubated with ab280086 for 30 mins at room temperature and followed by mouse specific IgG antibody (ab125913) for 8mins. The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used.

Heat mediated antigen retrieval with Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HeLa cells labelling SQSTM1 / p62 with ab280086 at 1/50 dilution, followed by ab150113 Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) antibody at 1/1000 dilution (Green). Confocal image showing cytoplasmic staining in HeLa cell line. ab179513 Anti-beta Tubulin rabbit monoclonal antibody was used to counterstain tubulin at 1/200 dilution followed by ab150080 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 594)(Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150113 Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) at 1/1000 dilution.

SQSTM1 / p62 was immunoprecipitated from 0.35 mg HeLa (human cervix adenocarcinoma epithelial cell ), whole cell lysate 10 ug with ab280086 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab280086 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: HeLa (human cervix adenocarcinoma epithelial cell ), whole cell lysate 10 ug

Lane 2: ab280086 IP in HeLa whole cell lysate

Lane 3: Mouse monoclonal IgG1(ab18443) instead of ab280086 in HeLa whole cell lysate

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

Exposure time: 26 seconds