Recombinant Anti-AMPK alpha 1 antibody [EPR24413-70] (ab271188)

Blocking and diluting buffer and concentration: Intercept® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS. Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

False colour image of Western blot: Anti-AMPK alpha 1 antibody [EPR24413-70] (ab271188) 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, ab271188 was shown to bind specifically to AMPK alpha 1. A band was observed at 63 kDa in wild-type HAP1 cell lysates with no signal observed at this size in AMPK alpha 1 knockout cell lysates. To generate this image, wild-type and AMPK alpha 1 knockout HAP1 cell lysates were analyzed. First, samples were run on an SDS-PAGE gel then transferred onto an immobilon-FL PVDF membrane. Membranes were blocked in Intercept®(TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS 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 1/10000 dilution.

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized Wild-type HAP1 (human chronic myelogenous leukemia near-haploid cell, Right) / PRAKK1 knockout HAP1(Left) cells labelling AMPK alpha 1 with ab271188 at 1/50 dilution (1μg) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat Anti-Rabbit IgG (Alexa Fluor^® 488, ab150081) at 1/2000 dilution was used as the secondary antibody.

Blocking and diluting buffer and concentration: Intercept^®(TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS. Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

False colour image of Western blot: Anti-AMPK alpha 1 antibody [EPR24413-70] (ab271188) 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, ab271188 was shown to bind specifically to AMPK alpha 1. A band was observed at 63 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 AMPK alpha 1 knockout RAW 264.7 cell lysates were analyzed. First, samples were run on an SDS-PAGE gel then transferred onto an immobilon-FL PVDF membrane. Membranes were blocked in Intercept^®(TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS 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 1/10000 dilution.

Immunohistochemical analysis of paraffin-embedded Mouse cerebrum tissue labelling AMPK alpha 1 with ab271188 at 1/100 (4.91 ug/ml) followed by a ready to use LeicaDS9800 (BOND™ Polymer Refine Detection). Positive staining on mouse cerebrum (PMID: 25538235) (PMID: 10098881). The section was incubated with ab271188 for 30 mins at room temperature.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 Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized Neuro-2a (Mouse neuroblastoma neuroblast) cells labelling AMPK alpha 1 with ab271188 at 1/50 dilution (1μg) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat Anti-Rabbit IgG (Alexa Fluor^® 488, ab150081) at 1/2000 dilution was used as the secondary antibody.

AMPK alpha 1 was immunoprecipitated from 0.35 mg Neuro-2a (mouse neuroblastoma neuroblast) whole cell lysate with ab271188 at 1/30 dilution (2 μg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab271188 at 1/2000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: Neuro-2a (mouse neuroblastoma neuroblast) whole cell lysate 10 μg

Lane 2: ab271188 IP in Neuro-2a whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab271188 in Neuro-2a whole cell lysate

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

Exposure time: 3 minutes

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

Lysates/proteins at 20 µg per lane.

Lane 4-7: This blot was developed using a higher sensitivity ECL substrate.

Exposure time: 3 minutes

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

This antibody has no cross-reaction with mouse AMPK alpha 2. Both recombinant proteins were made in-house and expressed from E.coli expression systems.

 Exposure time: 15 seconds

Immunohistochemical analysis of paraffin-embedded Rat cerebrum tissue labelling AMPK alpha 1 with ab271188 at 1/100 (4.91 μg/ml) followed by a ready to use LeicaDS9800 (BOND™ Polymer Refine Detection). Positive staining on rat cerebrum (PMID: 25538235) (PMID: 10098881). The section was incubated with ab271188 for 30 mins at room temperature.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 Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized C6 (rat glial tumor glial cell) cells labelling AMPK alpha 1 with ab271188 at 1/50 dilution (1μg) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat Anti-Rabbit IgG (Alexa Fluor^® 488, ab150081) at 1/2000 dilution was used as the secondary antibody.

AMPK alpha 1 was immunoprecipitated from 0.35 mg C6 (rat glial tumor glial cell) whole cell lysate with ab271188 at 1/30 dilution (2 μg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab271188 at 1/2000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: C6 (rat glial tumor glial cell) whole cell lysate 10 μg

Lane 2: ab271188 IP in C6 whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab271188 in C6 whole cell lysate

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

Exposure time: 3 minutes