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Abcam’s I?Bα (pS32/36) and I?Bα (Total) in vitro SimpleStep ELISA™ (Enzyme-Linked Immunosorbent Assay) kit is designed for the semi-quantitative measurement of I?Bα (pS32/36) and Total I?Bα protein in Human 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-I-κBα (Ser32/36): 0.06 ng/mL (tested with recombinant protein), Total I-κBα: 0.02 ng/mL (tested with recombinant protein)
Range: Phospho-I-κBα (Ser32/36): 0.06-6 ng/mL, Total I-κBα: 0.05-5 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 12ml|
|I kappaB alpha (pS32/36) Capture Antibody||1 x 1.5ml|
|I kappaB alpha (pS32/36) Detector Antibody||1 x 1.5ml|
|I kappaB alpha (Total) Capture Antibody||1 x 1.5ml|
|I kappaB alpha (Total) Detector Antibody||1 x 1.5ml|
|Lyophilized I kappaB alpha Control Lysate||1 vial|
|Plate Seal||1 vial|
|SimpleStep Pre-Coated 96-Well Microplate (ab206978)||1 vial|
|Stop Solution||1 x 12ml|
|TMB Substrate||1 x 12ml|
Our Abpromise guarantee covers the use of ab176661 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 IĸBα (pS32/36) and IĸBα (Total) cell lysate dilution series. Background-subtracted data values (mean +/- SD) are graphed.
Example of a typical IĸBα (pS32/36) and IĸBα (Total) recombinant protein standard curve. The proportion of total protein that is phosphorylated is unknown - data is indicative only. 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 IĸBα (pS32/36) are normalized and plotted.
Cell line analysis for Total IĸBα from 150 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 IĸBα (pS32/36) phosphorylation in HeLa 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 IĸBα (pS32/36) are plotted and compared against Total IĸBα protein levels. Comparative IĸBα (pS32/36) and IĸBα (Total) data also shown by Western Blot.
ab176661 has not yet been referenced specifically in any publications.
Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"