Anti-AMPK alpha 1 antibody [Y365]

12 product images

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  Western blot - 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] (Anti-GAPDH antibody [6C5] - Loading Control 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 Mouse PRKAA1 knockout RAW 264.7 cell line ab280055 (knockout cell lysate Mouse PRKAA1 knockout RAW 264.7 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 (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) at 1/20000 dilution.

  All lanes: Western blot - Anti-AMPK alpha 1 antibody [Y365] (ab32047) at 1/1000 dilution

  Lane 1: Wild-type RAW 264.7 cell lysate at 20 µg

  Lane 2: PRKAA1 knockout RAW 264.7 cell lysate at 20 µg

  Lane 2: Western blot - Mouse PRKAA1 knockout RAW 264.7 cell line (Mouse PRKAA1 knockout RAW 264.7 cell line ab280055)

  Lane 3: Mouse Liver cell lysate at 20 µg

  Lane 4: Neuro2A cell lysate at 20 µg

  Performed under reducing conditions.

  Predicted band size: 64 kDa

  Observed band size: 64 kDa

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

  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, Anti-GAPDH antibody [6C5] - 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 Anti-GAPDH antibody [6C5] - Loading Control 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 (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) secondary antibodies at 1/10,000 dilution for 1hr at room temperature before imaging.

  All lanes: Western blot - Anti-AMPK alpha 1 antibody [Y365] (ab32047)

  Predicted band size: 64 kDa

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-AMPK alpha 1 antibody [Y365] (ab32047)

  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. Goat Anti-Rabbit IgG H&L (HRP) 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.

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

  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 (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) instead of ab32047 in HeLa whole cell lysate.

  For western blotting, VeriBlot for IP Detection Reagent (HRP) 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.

  All lanes: Immunoprecipitation - Anti-AMPK alpha 1 antibody [Y365] (ab32047)

  Predicted band size: 64 kDa

  Observed band size: 63 kDa

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  Immunocytochemistry/ Immunofluorescence - Anti-AMPK alpha 1 antibody [Y365] (ab32047)

  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. Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) 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. Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291, a mouse anti-tubulin (1/1000) and Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/500) were also used.

  Control 1: primary antibody (1/250) and secondary antibody, Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/500).

  Control 2: Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291 (1/1000) and secondary antibody, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/500).

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  Flow Cytometry (Intracellular) - Anti-AMPK alpha 1 antibody [Y365] (ab32047)

  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.
  

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

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

  This antibody shows low sensitivity in mouse samples.

  All lanes: Western blot - Anti-AMPK alpha 1 antibody [Y365] (ab32047) at 1/500 dilution

  Lane 1: HepG2 (human liver hepatocellular carcinoma cell line) whole cell lysate at 20 µg

  Lane 2: NIH/3T3 (Mouse embryonic fibroblast) whole cell lysate at 20 µg

  Lane 3: Mouse liver tissue lysate at 20 µg

  Lane 4: Mouse brain tissue lysate at 20 µg

  Lane 5: Mouse kidney tissue lysate at 20 µg

  Lane 6: Mouse retina tissue lysate at 20 µg

  Lane 7: Mouse skeletal muscle tissue lysate at 20 µg

  Secondary

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

  Predicted band size: 64 kDa

  Observed band size: 40 kDa, 63 kDa

  Exposure time: 3min

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

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

  All lanes: Western blot - Anti-AMPK alpha 1 antibody [Y365] (ab32047) at 1/2000 dilution

  Lane 1: C6 cell lysate at 20 µg

  Lane 2: NIH/3T3 cell lysate at 20 µg

  Secondary

  All lanes: Peroxidase-conjugated goat anti-rabbit IgG, (H+L) at 1/1000 dilution

  Predicted band size: 64 kDa

  Observed band size: 63 kDa

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

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

  This antibody shows low sensitivity in mouse and rat samples.

  All lanes: Western blot - Anti-AMPK alpha 1 antibody [Y365] (ab32047) at 1/20000 dilution

  Lane 1: MCF7 (Human breast adenocarcinoma epithelial cell) whole cell lysate at 20 µg

  Lane 2: HepG2 (Human hepatocellular carcinoma epithelial cell) whole cell lysate at 20 µg

  Lane 3: HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate at 20 µg

  Lane 4: K-562 (Human chronic myelogenous leukemia lymphoblast) whole cell lysate at 20 µg

  Lane 5: C6 (Rat glial tumor glial cell) whole cell lysate at 20 µg

  Lane 6: Neuro-2a (Mouse neuroblastoma neuroblast) whole cell lysate at 20 µg

  Lane 7: NIH/3T3 (Mouse embryonic fibroblast) whole cell lysate at 20 µg

  Secondary

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

  Predicted band size: 64 kDa

  Observed band size: 63 kDa

  Exposure time: 180s

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  Flow Cytometry (Intracellular) - Anti-AMPK alpha 1 antibody [Y365] (ab32047)

  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) (Goat Anti-Rabbit IgG H&L (DyLight® 488) preadsorbed 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.
  

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-AMPK alpha 1 antibody [Y365] (ab32047)

  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.

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

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

  Anti-GAPDH antibody [EPR16891] - Loading Control ab181602 was used as a GAPDH loading control at 1/1000000 dilution.

  This blot was developed using a higher sensitivity ECL substrate.

  Compared with ab32047, Anti-AMPK alpha 1 antibody [EPR24413-70] ab271188 has higher sensitivity, we recommend Anti-AMPK alpha 1 antibody [EPR24413-70] ab271188 as an alternative for testing AMPK alpha 1 in mouse and rat samples in western blot.

  Lanes 1 - 8: Western blot - Anti-AMPK alpha 1 antibody [Y365] (ab32047) at 1/1000 dilution

  Lanes 1 - 8: Western blot - Anti-AMPK alpha 1 antibody [EPR24413-70] (Anti-AMPK alpha 1 antibody [EPR24413-70] ab271188) at 1/1000 dilution

  Lane 1: NIH/3T3 (mouse embryonic fibroblast) whole cell lysate at 20 µg

  Lane 2: Neuro-2a (mouse neuroblastoma neuroblast) whole cell lysate at 20 µg

  Lane 3: C6 (rat glial tumor glial cell) whole cell lysate at 20 µg

  Lane 4: Mouse brain tissue lysate at 20 µg

  Lane 5: Mouse heart tissue lysate at 20 µg

  Lane 6: Mouse skeletal muscle tissue lysate at 20 µg

  Lane 7: Rat brain tissue lysate at 20 µg

  Lane 8: Rat heart tissue lysate at 20 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution

  Developed using the ECL technique.

  Observed band size: 64 kDa

  Exposure time: 180s