Product nameAnti-AMPK alpha 1 (phospho T183) + AMPK alpha 2 (phospho T172) antibody
DescriptionRabbit polyclonal to AMPK alpha 1 (phospho T183) + AMPK alpha 2 (phospho T172)
Specificityab23875 recognises the phosphorylated forms of AMPK alpha 1 (T172) and AMPK alpha 2 (T183).
Tested applicationsSuitable for: WB, IHC-P, Flow Cyt, ICC/IFmore details
Species reactivityReacts with: Mouse, Rat, Human
Synthetic peptide corresponding to Human AMPK alpha 1 (phospho T183). Also within AMPK alpha 2 (phospho T172).
- Insulin treated CHO T cells, Insulin treated 3T3L1, Metformin treated L6 myoblast cells. Metformin treated HepG2 cells (10 mM for 24 hr).
Storage instructionsShipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C long term.
Storage bufferpH: 7.30
Preservative: 0.05% Sodium azide
Constituents: PBS, 50% Glycerol, 0.1% BSA
Concentration information loading...
PurityImmunogen affinity purified
Purification notesab23875 was purified from rabbit serum by sequential epitope specific chromatography. The antibody has been negatively preadsorbed using a non phosphopeptide corresponding to the site of phosphorylation to remove antibody that is reactive with non phosphorylated AMPK. The final product is generated by affinity chromatography using a AMPK derived peptide that is phosphorylated at threonine 172.
- Pathways and Processes
- Metabolic signaling pathways
- Lipid and lipoprotein metabolism
- Fatty acids
- Pathways and Processes
- Metabolic signaling pathways
- Energy transfer pathways
- Integration of energy
Our Abpromise guarantee covers the use of ab23875 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|WB||Use at an assay dependent concentration. Predicted molecular weight: 62 kDa.|
|IHC-P||1/10 - 1/50.|
Cellular localizationAMPK alpha 2: Cytoplasm. Nucleus. In response to stress, recruited by p53/TP53 to specific promoters.
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
Lane 6 : Lysates prepared from HepG2 cells stimulated with
Metformin and treated with lambda
phosphatase with 3% BSA-TBST buffer
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.
This product has been referenced in:
- Zhou T et al. Mst1 inhibition attenuates non-alcoholic fatty liver disease via reversing Parkin-related mitophagy. Redox Biol 21:101120 (2019). Read more (PubMed: 30708325) »
- Gao XL et al. Upregulation of microRNA-335-5p reduces inflammatory responses by inhibiting FASN through the activation of AMPK/ULK1 signaling pathway in a septic mouse model. Cytokine 110:466-478 (2018). Read more (PubMed: 29866515) »