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Synthetic peptide (the amino acid sequence is considered to be commercially sensitive) within Human AMPK alpha 1 aa 500 to the C-terminus (C terminal). The exact sequence is proprietary.
Database link: Q13131
Produced using Abcam's RabMAb® technology. RabMAb® technology is covered by the following U.S. Patents, No. 5, 675, 063 and/or 7, 429, 487.
We are constantly working hard to ensure we provide our customers with best in class antibodies. As a result of this work we are pleased to now offer this antibody in purified format. We are in the process of updating our datasheets. The purified format is designated 'PUR' on our product labels. If you have any questions regarding this update, please contact our Scientific Support team.
Our Abpromise guarantee covers the use of ab32047 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|Flow Cyt||1/100 - 1/150.
ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.
|WB||1/1000 - 1/5000. Predicted molecular weight: 63 kDa.|
|IHC-P||1/100 - 1/250. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
See protocols (link: http://www.abcam.com/protocols/ihc-antigen-retrieval-protocol).
|IP||1/40 - 1/50.|
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 knockout HAP1 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 when AMPK alpha knockout samples were used. 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 1 h 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. 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).
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.
Blocking and dilution buffer: 5% NFDM/TBST.
Blocking and dilution buffer: 5% NFDM/TBST.
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 (HRP) was used as the secondary antibody (1/1500).
Blocking buffer and concentration: 5% NFDM/TBST.
Diluting buffer and concentration: 5% NFDM /TBST.
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.
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.