{"product_id":"proteintech-66444-1-ig","title":"Proteintech, 66444-1-Ig, Phospho-AKT (Ser473) Monoclonal antibody","description":"Size: 20ul \/ 100ul\nThe Phospho-AKT (Ser473) (66444-1-Ig) by Proteintech is a Monoclonal antibody targeting Phospho-AKT (Ser473) in WB, IHC, IF\/ICC, FC (Intra), ELISA applications with reactivity to human, mouse, rat samples\n66444-1-Ig targets Phospho-AKT (Ser473) in WB, IHC, IF\/ICC, FC (Intra), ELISA applications and shows reactivity with human, mouse, rat samples.\n\u003cb\u003eTested Applications\u003c\/b\u003e\nPositive WB detected in: Calyculin A treated PC-3 cells, Calyculin A treated HEK-293T cells,  HSC-T6 cells,  TPA treated Jurkat cells,  Calyculin A treated HSC-T6 cells\nPositive IHC detected in: human breast cancer tissue, Calyculin A treated Jurkat cells,  human colon cancer tissueNote: suggested antigen retrieval withTE buffer pH 9.0;(*) Alternatively, antigen retrieval may be performed withcitrate buffer pH 6.0\nPositive IF\/ICC detected in: Calyculin A treated HeLa cells\nPositive FC (Intra) detected in: Calyculin A treated PC-3 cells\n\u003cb\u003eRecommended dilution\u003c\/b\u003e\nWestern Blot (WB): WB : 1:2000-1:10000\nImmunohistochemistry (IHC): IHC : 1:100-1:400\nImmunofluorescence (IF)\/ICC: IF\/ICC : 1:200-1:800\nFlow Cytometry (FC) (INTRA): FC (INTRA) : 0.50 ug per 10^6 cells in a 100 µl suspension\n\u003cb\u003eBackground Information\u003c\/b\u003e\n1) What is AKT?The serine\/threonine kinase B AKT pathway (also known as the PI3K-Akt pathway) plays a vital role in the regulation of cellular processes, including cell proliferation, survival, and growth - processes that are essential for oncogenesis. Mutation of the regulator proteins PI3K and PTEN causes uncontrolled disruption within the PI3-kinase pathway, leading to the development of human cancers (1,2; see alsoAKT pathway posterfor more details).2) phospho-AKT and FAQsA)  What is the best way to normalize phosphorylated proteins analyzed by western blot?Normalize phospho-AKT and total AKT with your loading control (e.g. Actin, tubulin), then calculate the phospho\/total ratio using these normalized values.Put more simply:1. Calculate the ratio of band intensities of a phospho-AKT band: the loading control.2. Calculate the ratio of band intensities of total AKT: loading control.3. Divide ratio obtained #1 by #2 to obtain a normalized value for comparison among different conditions. This procedure allows one to distinguish between a change in AKT expression and a change in the ratio of phospho-AKT.* If you are looking at the differences in a phospho-AKT expression resulting from an experimental condition (e.g., knockdown), you should also show the expression of total AKT to distinguish between a change in AKT expression (transcription\/translation level) and a change in the AKT phosphorylation status.B) What is the observed molecular weight for AKT and phospho-AKT?Molecular Weight AKT - 56 kDaMolecular Weight phospho-AKT - 60 kDa (Figure 1)Figure 1. WB: HEK-293 cell lysate was subjected to SDS PAGE followed by western blot with 60203-2-Ig (AKT antibody) and 66444-1-Ig (AKT-phospho-S473 antibody) at a dilution of 1:4000 incubated at room temperature for 1.5 hours.C) Are there any special WB conditions to optimize staining of a phospho-AKT?Since this is a phosphorylated protein, 5% BSA is recommended over non-fat milk as a blocking agent.D) What are good positive and negative controls for a phospho-AKT?- Positive Control: HEK293 cells- Negative Control: Treatment with PI3K inhibitors (e.g. wortmannin)E) What species does this antibody react with?Our internal testing has confirmed that it reacts with the human and mouse forms of phospho-AKT.Reactivity with the human form is also supported by theliterature's citations of this antibody.References:1.Perturbations of the AKT signaling pathway in human cancer.2.Targeting the PI3K-Akt pathway in human cancer: rationale and promise.\n\u003cb\u003eSpecification\u003c\/b\u003e\nTested Reactivity: human, mouse, rat\nCited Reactivity: human, mouse, rat, pig, rabbit, canine, monkey, chicken, bovine, duck\nHost \/ Isotype: Mouse \/ IgG1\nClass: Monoclonal\nType: Antibody\nImmunogen: Peptide Predict reactive species\nFull Name: v-akt murine thymoma viral oncogene homolog 1\nObserved Molecular Weight: 60-62 kDa\nGenBank Accession Number: NM_005163\nGene Symbol: AKT1\nGene ID (NCBI): 207\nRRID: AB_2782958\nConjugate: Unconjugated\nForm: Liquid\nPurification Method: Protein G purification\nUNIPROT ID: P31749\nStorage Buffer: PBS with 0.02% sodium azide and 50% glycerol, pH 7.3.\nStorage Conditions: Store at -20°C. Stable for one year after shipment. Aliquoting is unnecessary for -20 o C storage. 20ul sizes contain 0.1% BSA.\nFAQ\nA)  What is the best way to normalize phosphorylated proteins analyzed by western blot?Normalize phospho-AKT and total AKT with your loading control (e.g. Actin, tubulin), then calculate the phospho\/total ratio using these normalized values.Put more simply:1. Calculate the ratio of band intensities of a phospho-AKT band: the loading control.2. Calculate the ratio of band intensities of total AKT: loading control.3. Divide ratio obtained #1 by #2 to obtain a normalized value for comparison among different conditions. This procedure allows one to distinguish between a change in AKT expression and a change in the ratio of phospho-AKT.* If you are looking at the differences in a phospho-AKT expression resulting from an experimental condition (e.g., knockdown), you should also show the expression of total AKT to distinguish between a change in AKT expression (transcription\/translation level) and a change in the AKT phosphorylation status.B) What is the observed molecular weight for AKT and phospho-AKT?Molecular Weight AKT - 56 kDaMolecular Weight phospho-AKT - 60 kDa (Figure 1)\nFigure 1. WB: HEK-293 cell lysate was subjected to SDS PAGE followed by western blot with 60203-2-Ig (AKT antibody) and 66444-1-Ig (AKT-phospho-S473 antibody) at a dilution of 1:4000 incubated at room temperature for 1.5 hours.\nC) Are there any special WB conditions to optimize staining of a phospho-AKT?Since this is a phosphorylated protein, 5% BSA is recommended over non-fat milk as a blocking agent.D) What are good positive and negative controls for a phospho-AKT?- Positive Control: HEK293 cells- Negative Control: Treatment with PI3K inhibitors (e.g. wortmannin)E) What species does this antibody react with?\nOur internal testing has confirmed that it reacts with the human and mouse forms of phospho-AKT.Reactivity with the human form is also supported by theliterature's citations of this antibody.","brand":"Proteintech","offers":[{"title":"Default Title","offer_id":46887966900393,"sku":"66444-1-Ig","price":0.99,"currency_code":"USD","in_stock":true}],"url":"https:\/\/iright.com\/es\/products\/proteintech-66444-1-ig","provider":"Iright","version":"1.0","type":"link"}