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Abcam’s AKT 1/2/3 (pS473) and AKT1 (Total) in vitro SimpleStep™ ELISA (Enzyme-Linked Immunosorbent Assay) kit is designed for the semi-quantitative measurement of AKT 1/2/3 (pS473) and total AKT1 protein in Human and mouse cells.
The SimpleStep ELISA™ employs an affinity tag labeled capture antibody and a reporter conjugated detector antibody which immunocapture the sample analyte in solution. This entire complex (capture antibody/analyte/detector antibody) is in turn immobilized via immunoaffinity of an anti-tag antibody coating the well. To perform the assay, samples or standards are added to the wells, followed by the antibody mix. After incubation, the wells are washed to remove unbound material. TMB substrate is added and during incubation is catalyzed by HRP, generating blue coloration. This reaction is then stopped by addition of Stop Solution completing any color change from blue to yellow. Signal is generated proportionally to the amount of bound analyte and the intensity is measured at 450 nm. Optionally, instead of the endpoint reading, development of TMB can be recorded kinetically at 600 nm.
Estimated sensitivity: Phospho-AKT1/2/3 (Ser473): 0.5 ng/mL (tested with recombinant protein), Total AKT1: 0.2 ng/mL (tested with recombinant protein)
Range: Phospho-AKT1/2/3 (Ser473): 1-100 ng/mL, Total AKT1: 0.5-50 ng/mL
|Components||1 x 96 tests|
|10X Wash Buffer PT||1 x 15ml|
|50X Cell Extraction Enhancer Solution||1 x 1ml|
|5X Cell Extraction Buffer PTR (ab193970)||1 x 10ml|
|AKT 1/2/3 (pS473) Capture Antibody||1 x 1.5ml|
|AKT 1/2/3 (pS473) Detector Antibody||1 x 1.5ml|
|AKT1 (Total) Capture Antibody||1 x 1.5ml|
|AKT1 (Total) Detector Antibody||1 x 1.5ml|
|Lyophilized AKT Control Lysate||1 vial|
|Plate Seal||1 unit|
|SimpleStep Pre-Coated 96-Well Microplate (ab206978)||1 unit|
|Stop Solution||1 x 12ml|
|TMB Substrate||1 x 12ml|
Our Abpromise guarantee covers the use of ab176657 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|Sandwich ELISA||Use at an assay dependent concentration.|
Example of a typical AKT (pS473) and AKT1 (Total) cell lysate dilution series. Background-subtracted data values (mean +/- SD) are graphed.
Linearity of dilution in representative sample matrices. Cellular lysates were prepared at 3 concentrations in common media containing 1X Cell Extraction Buffer PTR. Data from duplicate measurements of AKT1 (pS473) are normalized and plotted.
Cell line analysis for total AKT1 from 20 µg/mL preparations of cell extracts. Data from triplicate measurements (mean +/- SD) are plotted and compared to 1X Cell Extraction Buffer PTR (zero).
Induction of AKT (pS473) phosphorylation in MCF-7 cells in response to insulin treatment. MCF-7 cells were cultured in 96-well tissue culture plates, serum-starved and treated (5 min) with a dose-range of insulin before cell lysis. Data from quadruplicate measurements of AKT (pS473) are plotted and compared against total AKT1 protein levels. Comparative AKT (pS473) and AKT1 (Total) data also shown by Western Blot.