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Abcam's NFkB p65 Transcription Factor Assay Kit (ab133112) is a non-radioactive, sensitive method for detecting specific transcription factor DNA binding activity in nuclear extracts.
A 96-well enzyme-linked immunosorbent assay (ELISA) replaces the cumbersome radioactive electrophoretic mobility shift assay (EMSA). A specific double stranded DNA (dsDNA) sequence containing the NFkB response element is immobilized onto the bottom of wells of a 96-well plate. NFkB contained in a nuclear extract, binds specifically to the NFkB response element. NFkB (p65) is detected by addition of specific primary antibody directed against NFkB (p65). A secondary antibody conjugated to HRP is added to provide a sensitive colorimetric readout at 450 nm.
|96-Well Plate Cover||1 unit|
|Polysorbate 20||1 vial|
|Transcription Factor Antibody Binding Buffer (10X)||1 x 3ml|
|Transcription Factor Binding Assay Buffer (4X)||1 x 3ml|
|Transcription Factor Developing Solution||1 x 12ml|
|Transcription Factor Goat Anti-Rabbit HRP Conjugate||1 x 100µl|
|Transcription Factor NFkB (Human p65) Positive Control||1 vial|
|Transcription Factor NFkB (p65) Primary Antibody||1 vial|
|Transcription Factor NF-kB 96-Well Strip Plate||1 unit|
|Transcription Factor NFkB Competitor dsDNA||1 vial|
|Transcription Factor Reagent A||1 x 120µl|
|Transcription Factor Stop Solution||1 x 12ml|
|Wash Buffer Concentrate (400X)||1 x 5ml|
Our Abpromise guarantee covers the use of ab133112 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|Functional Studies||Use at an assay dependent concentration.
It does not cross-react with NFkB (p50) transcription factor.
After the treatment with LPS (10 μg/ml for 6 hrs), cells were lysed with hypotonic HEPES lysis buffer (pH 7.4) and centrifuged at 1000 g for 10 min at 4°C, supernatants were collected and used for the determination of intracellular p65- NF-κB by ELISA. The absorbance was read at 450 nm using spectrophotometer.
Jurkat cells were treated with PMA and ionomycin (+). Nuclear lysates were extracted (ab113474) and 40 uL, corresponding to 4e6 cells, were tested in duplicates (+/- SD).
Titration of positive control with or without inhibitor, background signal subtracted (duplicates; +/- SD).