Immunofluorescence analysis of PRAS40 [pT246] was done by treating serum starved U-87 MG cells with 20 ng/ml PDGF for 15 minutes. 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™ PRAS40 [pT246] Recombinant Rabbit Monoclonal Antibody (701058) at a dilution of 1:400 in 1% BSA and incubated for 3 hours at room temperature and then labeled with Alexa Flour® 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 with DAPI (S36938). F-actin (Panel c: red) was stained with Alexa Fluor® 594 Phalloidin (A12381). Panel d is a merged image showing cytoplasmic and nuclear localization. Panel e shows competition with the phospho- PRAS40 [pT246] peptide. The images were captured using a Nikon microscope at 20X magnification.
|Tested species reactivity||Human, Mouse|
|Host / Isotype||Rabbit / IgG|
|Immunogen||Phosphopeptide corresponding to amino acids 241–251 of human PRAS40|
|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 *|
|Flow Cytometry (Flow)||1:100-1:500|
|Western Blot (WB)||1:500-1:5000|
* 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.
Proline Rich Akt Substrate of 40 kDa (PRAS40) is a signaling target known to play a regulatory role in growth, development, and apoptosis through the phosphoinositol kinase 3 pathway. PRAS40 also acts by binding to mTOR complex 1 and inhibiting mTOR function by altering nutrient balance, and growth maintenance. Phosphorylation of PRAS40 by mTORC1 at Serine 183, 212 and 221 residues disrupts this binding thus removing the inhibitory effect. Activated Akt phosphorylates PRAS40 on threonine 246. Mutation of PRAS40 threonine 246 has been shown to be apoptotic, and plays a role in abrogating neuronal protection.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.