Recombinant Anti-AMPK alpha 1 antibody [Y365] (ab32047)

False colour image of Western blot: Anti-AMPK alpha 1 antibody [Y365] staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab32047 was shown to bind specifically to AMPK alpha 1. A band was observed at 64 kDa in wild-type RAW 264.7 cell lysates with no signal observed at this size in PRKAA1 knockout cell line ab280055 (knockout cell lysate ab280114). To generate this image, wild-type and PRKAA1 knockout RAW 264.7 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged.Secondary antibodies used were Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: AMPK alpha knockout HAP1 cell lysate (20 µg)
Lane 3: MCF7 cell lysate (20 µg)
Lane 4: HeLa cell lysate (20 µg)
Lanes 1 - 4: Merged signal (red and green). Green - ab32047 observed at 63 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab32047 was shown to specifically react with AMPK alpha in wild-type HAP1 cells. No band was observed when AMPK alpha knockout samples were examined. Wild-type and AMPK alpha knockout samples were subjected to SDS-PAGE. ab32047 and ab8245 (loading control to GAPDH) were diluted 1/5000 and 1/2000 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 1hr at room temperature before imaging.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human lung carcinoma tissue labelling AMPK alpha 1 with purified ab32047 at 1/100. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9 before commencing with IHC staining protocol. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin. 

Immunocytochemistry/Immunofluorescence analysis of MCF7 cells labelling AMPK alpha 1 with purified ab32047 at 1/250. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/500) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain. ab7291, a mouse anti-tubulin (1/1000) and ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/500) were also used.

Control 1: primary antibody (1/250) and secondary antibody, ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/500).

Control 2: ab7291 (1/1000) and secondary antibody, ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/500).

Intracellular Flow Cytometry analysis of HeLa cells labelling AMPK alpha 1 with purified ab32047 at 1/150 (red). Cells were fixed with 2% paraformaldehyde. A FITC-conjugated goat anti-rabbit IgG (1/150) was used as the secondary antibody. Black - Isotype control, rabbit monoclonal IgG. Blue - Unlabelled control, cells without incubation with primary and secondary antibodies. 

ab32047 (purified) at 1/40 immunoprecipitating AMPK alpha 1 in HeLa whole cell lysate.

Lane 1 (input): HeLa whole cell lysate (10µg)

Lane 2 (+): ab32047 + HeLa whole cell lysate (10µg).

Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab32047 in HeLa whole cell lysate.

For western blotting, ab131366 VeriBlot for IP Detection Reagent (HRP) was used for detection (1/1500).

Blocking buffer and concentration: 5% NFDM/TBST.

Diluting buffer and concentration: 5% NFDM /TBST.

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

This antibody shows low affinity on mouse samples.

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

This antibody shows low affinity on mouse and rat samples.

Blocking and dilution buffer: 5% NFDM/TBST.
This antibody shows low affinity on mouse and rat samples.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analaysis of human cervical carcinoma tissue labelling AMPK alpha 1 with unpurified ab32047 at a dilution of 1/100. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Overlay histogram showing HeLa cells stained with unpurified ab32047 (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 (unpurified ab32047, 1/100 dilution) for 30 min at 22°C. The secondary antibody used was DyLight^® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1µg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.