Description
NFkB Phosphorylation Assay Kit (Fluorometric) (BA0136) (BA0136)
The NFkB Phosphorylation Assay Kit (Fluorometric) (SKU: BA0136) is a cell-based ELISA that measures the phosphorylation status of p65/RelA at serine 536 in whole cells. Nuclear factor-kappa B (NFkB) is a transcription factor central to many physiological processes including inflammation, tumorigenesis and apoptosis, and is activated by a wide variety of stimuli such as the inflammatory cytokine TNF-alpha. Phosphorylation of p65/RelA at Ser-536 results in decreased nuclear export and enhanced p65/RelA-dependent transcription. This assay measures phosphorylated p65(S536) in whole cells and normalises the signal to the total protein content, eliminating the need for cell lysate preparation and allowing the study of NFkB regulation in short-term and long-term assays. Protein phosphorylation is read fluorometrically at 530/585 nm and total protein at 360/450 nm. The total assay time has been reduced to 6.5 hours, with a hands-on time of 2.5 hours.
| Product Name: | NFkB Phosphorylation Assay Kit (Fluorometric) (BA0136) |
| SKU: | BA0136 |
| Detection Method: | Fluorometric cell-based ELISA |
| Sample Type: | Whole cells (adherent or suspension) |
| Species Reactivity: | Human, mouse |
| Assay Time: | 6.5 hours total (2.5 hours hands-on) |
| Kit Size: | 100 Assays |
| Equipment Required: | Microplate reader |
| Storage: | -20 degrees C |
| Shelf Life: | 6 months after receipt |
| Shipping: | Gel Pack |
This cell-based ELISA measures phosphorylated p65(S536) (pNFkB) in whole cells and normalises the signal to the total protein content in the same well. Cells are cultured directly in 96-well plates, and after fixation, quenching and blocking, a primary antibody against pNFkB p65(S536) and an HRP-conjugated secondary antibody are added, followed by fluorogenic substrates read at 530/585 nm for pNFkB and 360/450 nm for total protein.
- New and improved, with total assay time reduced from the standard 21 hours to 6.5 hours (hands-on time 2.5 hours)
- Simple and convenient, with cells cultured directly in 96-well plates and no cell lysis necessary
- Accurate and high-throughput, as protein phosphorylation is normalised to total cellular protein in the same well to minimise well-to-well variation and can be readily automated for thousands of samples per day
- Determination of NFkB p65(S536) phosphorylation status in whole cells
- Evaluation of direct and indirect modulation of NFkB p65 phosphorylation
Note: The below protocol is a sample protocol. Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.
| Step | Procedure |
| 1 | Prepare 1x Wash Buffer by diluting Stock Wash Buffer 40-fold with distilled water, reserving 6 mL for the detection step, and seed 100 uL of 1-3 x 10^4 adherent cells (or 4-10 x 10^4 suspension cells) into each well of a black 96-well culture plate, including culture media without cells for the Protein Blank, then incubate overnight at 37 degrees C. |
| 2 | Treat the cells as desired, then fix them by replacing the medium with 100 uL of 4% formaldehyde (adherent cells) or adding 8% formaldehyde to the cell pellet (suspension cells) and incubate 20 minutes at room temperature. |
| 3 | Wash the cells three times with 150 uL of 1x Wash Buffer, add 100 uL of Quench Buffer (2.2 mL 3% H2O2 in 8.8 mL 1x Wash Buffer) and incubate 20 minutes at room temperature, then wash three times and add 100 uL Blocking Buffer for 1 hour at room temperature. |
| 4 | Add 50 uL of Ab1 Mixture (Ab1 diluted 1:625 in Blocking Buffer) to the sample wells and 50 uL Blocking Buffer to the sample blank wells, incubate 90 minutes at room temperature (or overnight at 2-8 degrees C), then wash three times. |
| 5 | Add 50 uL of Ab2 Mixture (Ab2 diluted 1:625 in Blocking Buffer) to all assay wells, incubate 90 minutes at room temperature with gentle shaking, then wash five times. |
| 6 | Add 50 uL of freshly prepared HRP Substrate (60 uL Dye Reagent, 6 mL 1x Wash Buffer, 6 uL 3% H2O2) to each well and incubate 30 minutes in the dark, then add 50 uL Protein Stain and incubate a further 5 minutes in the dark. |
| 7 | Read the plate at 530/585 nm for phosphorylated NFkB and at 360/450 nm for total protein. |
Calculate the mean pNFkB fluorescence at 530/585 nm for the Sample Blank and Sample wells, and the mean protein fluorescence at 360/450 nm for the Protein Blank and Sample wells. Subtract the Sample Blank pNFkB fluorescence from the Sample pNFkB fluorescence to yield delta FpNFkB, and subtract the Protein Blank fluorescence from the Sample protein fluorescence to yield delta FProt. Normalised pNFkB = (delta FpNFkB / delta FProt) / (delta FpNFkB / delta FProt)o, where the denominator is the control reference value (e.g. time zero in kinetic studies or untreated wells in drug potency studies).
| Component | Quantity | Storage |
| Stock Wash Buffer | 25 mL | -20 degrees C |
| Blocking Buffer | 25 mL | -20 degrees C |
| Protein Stain | 6 mL | -20 degrees C |
| Dye Reagent | 120 uL | -20 degrees C |
| Ab1 | 10 uL | -20 degrees C |
| Ab2 (gR-HRP) | 10 uL | -20 degrees C |
