Immunofluorescence analysis of AMPK alpha [pT172] was done on 70% confluent log phase HeLa 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 ABfinity™ AMPK alpha [pT172] Recombinant Rabbit Oligoclonal Antibody (710099) at 3 in 1% BSA and incubated for 3 hours at room temperature and then labeled with Alexa Fluor 488 Goat Anti-Rabbit IgG Secondary Antibody (A11008) at a dilution of 1:400 for 30 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant DAPI (S36938). F-actin (Panel c: red) was stained with Alexa Fluor 594 Phalloidin (A12381). Panel d is a merged image showing nuclear localization. Panel e shows no primary antibody control. The images were captured at 20X magnification.
|Tested species reactivity||Human, Non-human primate|
|Host / Isotype||Rabbit / IgG|
|Immunogen||Peptide 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)||2-4 ug/ml|
|Immunofluorescence (IF)||2-4 ug/ml|
|Western Blot (WB)||1-3 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.
This antibody is predicted to react with mouse, rat, non-human primate and Drosophila based on sequence homology.
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™ oligoclonal antibodies comprise a selection of multiple different recombinant monoclonal antibodies, providing the best of both worlds—the sensitivity of a polyclonal antibody with the specificity of a monoclonal, all delivered with the consistency only found in a recombinant antibody. While functionally the same as a polyclonal antibody—recognizing multiple epitope sites on the target and producing higher detection sensitivity for low abundance targets when compared with monoclonal antibodies—an oligoclonal antibody has a known mixture of light and heavy chains. This exact population can be produced in every lot, circumventing the biological variability typically associated with polyclonal antibody production.
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.