Proteintech, 66444-1-Ig, Phospho-AKT (Ser473) Monoclonal antibody

Size: 20ul / 100ul The 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 66444-1-Ig targets Phospho-AKT (Ser473) in WB, IHC, IF/ICC, FC (Intra), ELISA applications and shows reactivity with human, mouse, rat samples. Tested Applications Positive 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 Positive 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 Positive IF/ICC detected in: Calyculin A treated HeLa cells Positive FC (Intra) detected in: Calyculin A treated PC-3 cells Recommended dilution Western Blot (WB): WB : 1:2000-1:10000 Immunohistochemistry (IHC): IHC : 1:100-1:400 Immunofluorescence (IF)/ICC: IF/ICC : 1:200-1:800 Flow Cytometry (FC) (INTRA): FC (INTRA) : 0.50 ug per 10^6 cells in a 100 µl suspension Background Information 1) 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. Specification Tested Reactivity: human, mouse, rat Cited Reactivity: human, mouse, rat, pig, rabbit, canine, monkey, chicken, bovine, duck Host / Isotype: Mouse / IgG1 Class: Monoclonal Type: Antibody Immunogen: Peptide Predict reactive species Full Name: v-akt murine thymoma viral oncogene homolog 1 Observed Molecular Weight: 60-62 kDa GenBank Accession Number: NM_005163 Gene Symbol: AKT1 Gene ID (NCBI): 207 RRID: AB_2782958 Conjugate: Unconjugated Form: Liquid Purification Method: Protein G purification UNIPROT ID: P31749 Storage Buffer: PBS with 0.02% sodium azide and 50% glycerol, pH 7.3. Storage 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. FAQ A)  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.