Immunofluorescence analysis of AMPKA (pT172) was done on 70% confluent log phase MDA-MB-231 cells. 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 Phospho-AMPK alpha-1 pThr172 (10H2L20), ABfinity™ Rabbit Monoclonal Antibody (701068) at 1ug/mL in 1% BSA and incubated for 3 hours at room temperature and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (S36938). F-actin (Panel c: red) was stained with Alexa Fluor® 555 Rhodamine Phalloidin (Product # R415, 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.
|Tested species reactivity||Human|
|Host / Isotype||Rabbit / IgG|
|Immunogen||Phosphopeptide corresponding to amino acids 168–178 of human AMPK alpha|
|Contains||0.09% sodium azide|
|Storage Conditions||Maintain refrigerated at 2-8°C for up to 1 month. For long term storage store at -20°C|
|Tested Applications||Dilution *|
|Immunocytochemistry (ICC)||1 µg/ml|
|Immunofluorescence (IF)||1 µg/ml|
|Western Blot (WB)||2-5 ug/ml|
* Suggested working dilutions are given as a guide only. It is recommended that the user titrate the product for use in their own experiment using appropriate negative and positive controls.
ABfinity™ recombinant antibodies are rabbit monoclonal antibodies, unmatched for producing superior results. ABfinity™ antibodies are developed by immunizing animals, screening for functionality, cloning the immunogen-specific antibody genes into high-level mammalian expression vectors, produced on a large scale, and purified with Protein A.
ABfinity™ monoclonal antibodies resemble rabbit monoclonals isolated from serum or produced by hybridomas, but demonstrate greater specificity and sensitivity. Because ABfinity™ recombinant antibodies are derived from cloned DNA sequences of the heavy and light antibody chains, they are not susceptible to cell-line drift or lot-to-lot variation, thus allowing for peak specificity and performance.
Intact IgG appears on a non-reducing gel as ~150 kDa band and upon reduction generating a ~25 kDa light chain band and a ~50 kDa heavy chain.
AMPKa belongs to the family of AMP activated protein kinases, that plays a central role in regulating cellular and organismal energy balance in response to the balance between AMP/ATP, and intracellular Ca2+ levels. AMPK exists as a heterotrimer comprising of catalytic alpha subunit, and regulatory beta and gamma subunits. The alpha subunit is activated by increases in the cellular AMP:ATP ratio caused by metabolic stresses, and can mediate the effects of the adipokines leptin and adiponectin. Two isoforms differ in subcellular localization and AMP-dependence. Phosphorylation of threonine 172 in the alpha subunit is a key determinant of AMPK activity.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.