Immunofluorescence analysis of AKT [pS473] was done on 70% confluent log phase U-87 MG cells. The cells were fixed with 4% paraformaldehyde for 15 minutes; permeabilized with 0.25% Triton™ X-100 for 10 minutes followed by blocking with 5% BSA for 1 hour at room temperature. The cells were incubated with ABfinity™ AKT [pS473] Recombinant Rabbit Monoclonal Antibody (700392) at 1 µg-2 µg 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 localization of AKT [pS473]. Panel e shows competition with AKT [pS473] peptide. The images were captured at 20X magnification.
|Tested species reactivity||Human, Mouse|
|Published species reactivity||Rat, Human, Not Applicable|
|Host / Isotype||Rabbit / IgG|
|Immunogen||A peptide corresponding to amino acids 468-477 of P31749.|
|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 *|
|ELISA (ELISA)||1-5 ug/ml|
|Flow Cytometry (Flow)||1-3µg/10^6 cells|
|Immunohistochemistry (Paraffin) (IHC (P))||0.5-2 ug/ml|
|Western Blot (WB)||1-3µg/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 bovine, canine, chicken, chimpanzee, equine, feline, rat, Rhesus monkey , Xenopus and zebrafish 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™ 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.
AKT also known as protein kinase B (PKB) or RAS-alpha, is an ubiquitous serine/threonine kinase that plays an important role in diverse biological responses such as regulation of metabolism, cell survival and growth by phosphorylating multiple proteins. This protein kinase is activated by insulin, PI3K, IGF1 and various other growth and survival factors. Akt promotes cell survival by inhibiting apoptosis through phosphorylation and inactivation of several targets, including forkhead transcription factors, and caspase-9. The AKT pathway is a major target for cancer drug discovery.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.
|Not Applicable||Not Cited||
Synergistic Activity of Deguelin and Fludarabine in Cells from Chronic Lymphocytic Leukemia Patients and in the New Zealand Black Murine Model.
700392 was used in flow cytometry to study the synergistic activity of fludarabine and deguelin in chronic lymphocytic leukemia patient cells in the New Zealand Black Murine model
|Rebolleda N,Losada-Fernandez I,Perez-Chacon G,Castejon R,Rosado S,Morado M,Vallejo-Cremades MT,Martinez A,Vargas-Nuñez JA,Perez-Aciego P||PloS one (11:null)||2016|
Snail heterogeneity in clear cell renal cell carcinoma.
700392 was used in immunohistochemistry - paraffin section to study clear cell renal cell carcinoma and snail heterogeneity
|Zaldumbide L,Erramuzpe A,Guarch R,Pulido R,Cortés JM,López JI||BMC cancer (16:null)||2016|
|Not Applicable||Not Cited||
Harnessing Connectivity in a Large-Scale Small-Molecule Sensitivity Dataset.
700392 was used in western blot to learn the connections between sensitivity in a small-molecule dataset
|Seashore-Ludlow B,Rees MG,Cheah JH,Cokol M,Price EV,Coletti ME,Jones V,Bodycombe NE,Soule CK,Gould J,Alexander B,Li A,Montgomery P,Wawer MJ,Kuru N,Kotz JD,Hon CS,Munoz B,Liefeld T,Dan¿ík V,Bittker JA,Palmer M,Bradner JE,Shamji AF,Clemons PA,Schreiber SL||Cancer discovery (5:1210)||2015|
Intramembrane binding of VE-cadherin to VEGFR2 and VEGFR3 assembles the endothelial mechanosensory complex.
700392 was used in western blot to study the role of VE-cadherin in fluid shear stress
|Coon BG,Baeyens N,Han J,Budatha M,Ross TD,Fang JS,Yun S,Thomas JL,Schwartz MA||The Journal of cell biology (208:975)||2015|
|Rat||1:1000||Nanoparticle-mediated signaling endosome localization regulates growth cone motility and neurite growth.||Steketee MB,Moysidis SN,Jin XL,Weinstein JE,Pita-Thomas W,Raju HB,Iqbal S,Goldberg JL||Proceedings of the National Academy of Sciences of the United States of America (108:19042)||2011|
Expression of EGFR and molecules downstream to PI3K/Akt, Raf-1-MEK-1-MAP (Erk1/2), and JAK (STAT3) pathways in invasive lung adenocarcinomas resected at a single institution.
700392 was used in immunohistochemistry (paraffin) to analyze retrospectively the protein expression of EGFR, Stat3, phospho-Akt, and phospho-Erk1/2 in tumors.
|Torres AF,Nogueira C,Magalhaes J,Costa IS,Aragao A,Gomes Neto A,Martins F,Tavora F||Analytical cellular pathology (Amsterdam) (2014:null)||2015|