Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human bladder carcinoma tissue sections labeling AMPK beta 1 with purified ab32112 at 1:1000 dilution (0.85 μg/ml). Heat mediated antigen retrieval was performed using Perform heat mediated antigen retrieval using EDTA Buffer, pH9.0. Tissue was counterstained with Hematoxylin. ImmunoHistoProbe one step HRP Polymer (ready to use) secondary antibody was used at 1:0 dilution. PBS instead of the primary antibody was used as the negative control.

All lanes : Anti-AMPK beta 1 antibody [Y367] (ab32112) at 1/5000 dilution (purified)

Lane 1 : Mouse brain lysates
Lane 2 : Rat brain lysates

Lysates/proteins at 15 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 30 kDa
Observed band size: 38 kDa why is the actual band size different from the predicted?

Blocking and diluting buffer: 5% NFDM/TBST

Flow Cytometry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling AMPK beta 1 with purified ab32112 at 1:800 dilution (1 ug/ml) (red). Cells were fixed with 80% methanol. A Goat anti rabbit IgG (Alexa Fluor^® 488) secondary antibody was used at 1:2000 dilution. Isotype control - 0.1% Tween-20. Unlabeled control - Rabbit monoclonal IgG (Black).

ab32112 (purified) at 1:40 dilution (2ug) immunoprecipitating AMPK beta 1 in NIH/3T3 (Mouse embryonic fibroblast) whole cell lysates.

Lane 1 (input): NIH/3T3 (Mouse embryonic fibroblast) whole cell lysates 10ug

Lane 2 (+): ab32112 & NIH/3T3 (Mouse embryonic fibroblast) whole cell lysates

Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab32112 in NIH/3T3 (Mouse embryonic fibroblast) whole cell lysates

For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366) was used for detection at 1:1000 dilution.

Blocking and diluting buffer: 5% NFDM/TBST.

Anti-AMPK beta 1 antibody [Y367] (ab32112) at 1/20000 dilution (purified) + HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates at 20 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 30 kDa
Observed band size: 38 kDa why is the actual band size different from the predicted?

Blocking and diluting buffer: 5% NFDM/TBST

All lanes : Anti-AMPK beta 1 antibody [Y367] (ab32112) at 1/5000 dilution (purified)

Lane 1 : NIH/3T3 (Mouse embryonic fibroblast) whole cell lysates
Lane 2 : PC-12 (Rat adrenal gland pheochromocytoma ) whole cell lysates

Lysates/proteins at 15 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 30 kDa
Observed band size: 38 kDa why is the actual band size different from the predicted?

Blocking and diluting buffer: 5% NFDM/TBST

Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: AMPK beta 1 knockout HAP1 cell lysate
Lane 3: HeLa cell lysate
Lane 4: A431 cell lysate
Lanes 1 - 4: Merged signal (red and green). Green - Unpurified ab32112 observed at 38 kDa. Red - loading control, ab18058, observed at 124 kDa.

Unpurified ab32112 was shown to specifically react with AMPK beta 1 when AMPK beta 1 knockout samples were used. Wild-type and AMPK beta 1 knockout samples were subjected to SDS-PAGE. ab32112 and ab18058 (loading control to Vinculin) were both diluted 1/10000 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 1/10000 dilution for 1 hour at room temperature before imaging.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human colon tissue sections labeling AMPK beta 1 with purified ab32112 at 1:1000 dilution (0.85 μg/ml). Heat mediated antigen retrieval was performed using Perform heat mediated antigen retrieval using EDTA Buffer, pH9.0. Tissue was counterstained with Hematoxylin. ImmunoHistoProbe one step HRP Polymer (ready to use) secondary antibody was used at 1:0 dilution. PBS instead of the primary antibody was used as the negative control.

All lanes : Anti-AMPK beta 1 antibody [Y367] (ab32112) at 1/5000 dilution (unpurified)

Lane 1 : (A) : NIH 3T3.
Lane 2 : (B) : Hela.
Lane 3 : (C) : A431.
Lane 4 : (D) : PC-12.

Predicted band size: 30 kDa
Observed band size: 38 kDa why is the actual band size different from the predicted?

Immunocytochemistry/Immunofluorescence analysis of MCF-7 cells labelling AMPK beta 1 with purified ab32112 at 1/500. Cells were fixed with 4% Paraformaldehyde and permeabilised with 0.1% tritonX-100. An Alexa Fluor® 488-conjugated goat anti-rabbit IgG (ab150077) at 1/1000 dilution was used as the secondary antibody. Nuclei counterstained with DAPI (blue).

Secondary Only Control: PBS was used instead of the primary antibody as the negative control.

Unpurified ab32112 at a 1:100 dilution staining AMPK beta 1 in human lung carcinoma, using Immunohistochemistry, Paraffin Embedded Tissue.

Overlay histogram showing HeLa cells stained with unpurified ab32112 (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 (ab32112, 1/100 dilution) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was rabbit IgG (monclonal) (1µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in HeLa cells fixed with 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.