NFκB Phosphorylation Status Screening Service

Summary

Using our EnzyFluo™ NFκB Phosphorylation Assay Kit, we screen for modulators that increase or decrease NFκB phosphorylation status in cells. We measure the NFκB phosphorylation kinetics, EC₅₀, and IC₅₀ of cells treated with modulators. Testing can be run on a wide variety of cell lines.

Species Reactivity

We first determine the test compound’s reactivity with different species. In this example, the species tested were human (MDA-MB-231), mouse (NIH-3T3), and rat (RBL-2H3). Here we use Tumor necrosis factor alpha (TNF-α) as an example test compound. In a sterile, black 96-well plate, MDA-MB-231, NIH-3T3, and RBL-2H3 cells were plated at a cell density of 20,000 cells per well and allowed to adhere overnight. The next day, the culture medium was removed and replaced with medium dosed with 10 ng/mL TNF-α or no TNF-α for the baseline controls. The dosed culture medium was prepared from a stock solution of 10,000 ng/mL TNF-α in dH2O. Triplicate wells were incubated for 10 minutes with the TNF-α or control medium. The pNFκB and total protein was then quantified using the NFκB Phosphorylation Assay Kit (EFNKB-100). The pNFκB values were normalized to the total protein content of the sample.

The relative increase in pNFκB for the cells treated with TNF-α was 3.64:1, 2.96:1, and 0.83:1 for MDA-MB-231, NIH-3T3, and RBL-2H3 respectively. The cell line MDA-MB-231 showed the greatest reactivity to the test compound TNF-α and was chosen as the cell line for the next experiments.

Phosphorylation Titration

To determine the time course of NFκB phosphorylation, first a titration was run on the concentration of the test compound; here we use TNF-α as an example. In a sterile, black 96-well plate, MDA-MB-231 cells were plated at a cell density of 20,000 cells per well and allowed to adhere overnight. The next day, the culture medium was removed and replaced with medium dosed with TNF-α at the following concentrations: 0.0E+0, 3.8E-4, 1.5E-3, 6.1E-3, 2.4E-2, 9.8E-2, 3.9E-1, 1.6E+0, 6.3E+0, 2.5E+1, 1.0E+2, and 4.0E+2 ng/mL. The dosed culture medium was prepared from a stock solution of 10,000 ng/mL TNF-α in dH2O. Triplicate wells were incubated for 20 minutes with the TNF-α. The pNFκB and total protein was then quantified using the NFκB Phosphorylation Assay Kit (ENFKB-100). The pNFκB values were normalized to the total protein content of the sample.

Phosphorylation Kinetics

After the titration on the test compound concentration, we measure the NFκB phosphorylation kinetics for a specific dosage. TNF-α will continue to serve as our example here; we measure the NFκB phosphorylation kinetics for TNF-α at a concentration of 100 ng/mL. In a sterile, black 96-well plate, PANC-1 cells were plated at a cell density of 20,000 cells per well and allowed to adhere overnight. The next day, the culture medium was removed and replaced with 100 ng/mL TNF-α dosed medium. The dosed culture medium was prepared from a stock solution of 10,000 ng/mL TNF-α in dH2O. Triplicate wells were incubated for 0, 2, 4, 6, 8, 10, 15, 20, 30, 40, 60, and 120 minutes with the TNF-&alpha. The pNFκB and total protein was then quantified using the NFκB Phosphorylation Assay Kit (ENFKB-100). The pNFκB values were normalized to the total protein content of the sample. The 0 minutes (no TNF-α incubation) served as the untreated control for basal NFκB phosphorylation.

Please email or call 1-510-782-9988 x 2 to request assay service.

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