All lanes : Anti-AMPK alpha 1 (phospho T183) + AMPK alpha 2 (phospho T172) antibody (ab23875)

Lane 1 : Lysates prepared from HepG2 cells left unstimulated with 3% BSA-TBST buffer and no peptide
Lane 2 : Lysates prepared from HepG2 cells stimulated with
Metformin with 3% BSA-TBST buffer and no peptide
Lane 3 : Lysates prepared from HepG2 cells stimulated with
Metformin with 3% BSA-TBST buffer and the non-phosphopeptide corresponding to the immunogen
Lane 4 : Lysates prepared from HepG2 cells stimulated with
Metformin with 3% BSA-TBST buffer and a
generic phospho-threonine-containing peptide
Lane 5 : Lysates prepared from HepG2 cells stimulated with
Metformin with 3% BSA-TBST buffer and the phosphopeptide
immunogen
Lane 6 : Lysates prepared from HepG2 cells stimulated with
Metformin and treated with lambda
phosphatase with 3% BSA-TBST buffer

Secondary
All lanes : goat F(ab’)2 anti rabbit
IgG HRP conjugate

Predicted band size: 62 kDa
Observed band size: 60 kDa why is the actual band size different from the predicted?


Exposure time: 2 hours

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.

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.

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.

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.