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Abcam’s NF?B p65 in vitro SimpleStep ELISA™ (Enzyme-Linked Immunosorbent Assay) kit is designed for the semi-quantitative measurement of NF?B p65 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.
|Components||1 x 96 tests|
|10X NF kappaB p65 (Total) Capture Antibody||1 x 3ml|
|10X NF kappaB p65 (Total) Detector Antibody||1 x 3ml|
|10X Wash Buffer PT||1 x 15ml|
|50X Cell Extraction Enhancer Solution||1 x 1ml|
|5X Cell Extraction Buffer PTR||1 x 10ml|
|Lyophilized NF kappaB p65 Control Lysate||1 vial|
|Plate Seal||1 unit|
|SimpleStep Pre-Coated 96 Well Microplate (12 x 8 well strips)||1 unit|
|Stop Solution||1 x 12ml|
|TMB Substrate||1 x 12ml|
Our Abpromise guarantee covers the use of ab176648 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 NFĸB p65 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 1 x Cell Extraction Buffer PTR. Data from duplicate measurements of NFĸB p65 (Total) are normalized and plotted.
Cell line analysis for Total NFĸB p65 from 200 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 NFĸB p65 (pS536) phosphorylation in MCF-7 cells in response to TNFα treatment. HeLa cells were cultured in 96-well tissue culture plates, and treated (10 min) with a dose-range of TNFα before cell lysis. Data from quadruplicate measurements of NFĸB p65 (pS536) are plotted and compared against total NFĸB p65 protein levels. Comparative NFĸB p65 (pS536) and NFĸB p65 (Total) data also shown by Western Blot.
ab176648 has not yet been referenced specifically in any publications.