Anti-AMPK alpha 1 (phospho T183) + AMPK alpha 2 (phospho T172) antibody

• WB

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Western blot - Anti-AMPK alpha 1 (phospho T183) + AMPK alpha 2 (phospho T172) antibody (AB23875)

Lysates were resolved by SDS-PAGE on a 10% polyacrylamide gel and transferred to PVDF, treated or not with lambda phosphatase, blocked with a 3% BSA-TBST buffer for one hour at room temperature, incubated with relevant peptides (see below) and incubated with the AMPK alpha 1/2 [pT 172] antibody for two hours at room temperature in 3% BSA-TBST buffer.

After washing, membranes were incubated with goat F(ab')2 antirabbit IgG HRP conjugate and bands were detected using the Pierce SuperSignal™ method.

All lanes:

Western blot - Anti-AMPK alpha 1 (phospho T183) + AMPK alpha 2 (phospho T172) antibody (ab23875)

Lane 1:

Lysates prepared from HepG2 cells left unstimulated

Lane 2:

Lysates prepared from HepG2 cells stimulated with Metformin

Lane 3:

Lysates prepared from HepG2 cells stimulated with Metformin with non-phosphopeptide corresponding to the immunogen

Lane 4:

Lysates prepared from HepG2 cells stimulated with Metformin with ageneric phospho-threonine-containing peptide

Lane 5:

Lysates prepared from HepG2 cells stimulated with Metformin with phosphopeptide immunogen

Lane 6:

Lysates prepared from HepG2 cells stimulated with Metformin and treated with lambdaphosphatase

Secondary

All lanes:

goat F(ab’)2 anti rabbitIgG HRP conjugate

Observed band size: 60 kDa

false

Exposure time: 1min

• Flow Cyt

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Flow Cytometry - Anti-AMPK alpha 1 (phospho T183) + AMPK alpha 2 (phospho T172) antibody (AB23875)

Flow Cytometry analysis of MDA-MB-231 cells labeling AMPK alpha 1 (phospho T183) + AMPK alpha 2 (phospho T172) with ab23875. Cells were fixed with 70% ethanol for 10 minutes, permeabilized with 0.25% Triton™ X-100 for 20 minutes, and blocked with 5% BSA for 30 minutes at room temperature. Cells were labeled with Anti-AMPK alpha 1 (phospho T183) + AMPK alpha 2 (phospho T172) antibody (ab23875, red) or with rabbit isotype control (pink) at 3-5 ug/million cells in 2.5% BSA. After incubation at room temperature for 2 hours, the cells were labeled with Alexa Fluor^® 488 Goat Anti-Rabbit Secondary Antibody at a dilution of 1/400 for 30 minutes at room temperature. The representative 10,000 cells were acquired and analyzed for each sample using an Attune^® Acoustic Focusing Cytometer. The purple histogram represents unstained control cells and the green histogram represents no-primary-antibody control.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-AMPK alpha 1 (phospho T183) + AMPK alpha 2 (phospho T172) antibody (AB23875)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human brain tissue sections labeling AMPK alpha 1 (phospho T183) + AMPK alpha 2 (phospho T172) with ab23875 (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H₂O₂-methanol for 15 min at room temperature, washed with ddH₂O and PBS, and then probed with AMPK alpha 1 (phospho T183) + AMPK alpha 2 (phospho T172) antibody (ab23875) diluted in 3% BSA-PBS at a dilution of 1/20 overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-AMPK alpha 1 (phospho T183) + AMPK alpha 2 (phospho T172) antibody (AB23875)

Immunocytochemistry/ Immunofluorescence analysis of 70% confluent log phase MDA-MB-231 cells labeling AMPK alpha 1 (phospho T183) + AMPK alpha 2 (phospho T172) with ab23875. The cells were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.25% Triton™ X-100 for 10 minutes, and blocked with 5% BSA for 1 hour at room temperature. The cells were labeled with Anti-AMPK alpha 1 (phospho T183) + AMPK alpha 2 (phospho T172) antibody (ab23875) at 1ug/mL in 1% BSA and incubated for 3 hours at room temperature and then labeled with Goat anti-Rabbit IgG (H+L) secondary antibody, Alexa Fluor^® 488 conjugate at a dilution of 1/2000 for 45 minutes at room temperature (Panel a : green). Nuclei (Panel b : blue) were stained with mountant with DAPI. F-actin (Panel c : red) was stained with Alexa Fluor^® 555 Rhodamine Phalloidin, 1/300. Panel d is a merged image showing Nuclear localization. Panel e is a no primary antibody control. The images were captured at 60X magnification.