Background
Members of the Rel/NF-kB family of transcription factors form one of the first lines of defense against infectious diseases and cellular stress (immune response). These proteins initiate a highly coordinated response in multiple cell types that effectively counteract the threat to the health of the organism. Activation of NF-kB has been reported to occur in response to a wide variety of stimuli such as cytokines, growth factors, physiological, physical and oxidative stress and certain pharmaceutical drugs and chemicals. These stimuli generally target the protein IkB to which the two major subunits of NF-kB, p50 and p65 are bound, to form its inactive form in the cytoplasm. Phosphorlyation and ubiquitination of IkB releases NF-kB, allowing it to translocate into the nucleus as the activated form.

Assay Principle
This chemiluminescence based sandwich type ELISA employs an oligonucleotide, containing the DNA binding NF-kB consensus sequence, bound to a 96-well formatted ELISA plate. The transcription factor, NF-kB present in the sample (cell/lymphocyte extracts) binds specifically to the oligonucleotide coated on the plate. The DNA bound NF-kB is selectively recognized by the primary antibody, which in turn is picked up by the secondary antibody-alkaline phosphatase conjugate. The Relative Light Units (RLU) measured by a chemiluminescence detector, after addition of alkaline phosphatase substrate, offer greater sensitivity than absorbance detection. This degree of sensitivity coupled by the linear correlation (r2 typically 0.99) between RLUs and amount of NF-kB allows precise and quantitative measurement of the transcription factor in biological samples using a standard curve.

Our protocol allows for successful quantitation (pg NF-kB /µg protein) of both the activated form of NF-kB, which is translocated into the nucleus and the total amount of NF-kB present in the cytoplasm as the complexed dormant form. Figure 2 shows a quantitative increase in relative light units with increase in protein concentrations in human lymphocytes treated with two different buffer systems. Thus both the activated or nuclear (open circles; r2 = 0.99) NF-kB and total (closed circles; r2 = 0.98) NF-kB present in the cytoplasm may be measured quantitatively, depending on ones application. Typically, there is an eight to ten fold difference between the activated and total amount of NF-kB (pg/µg protein) present in human lymphocytes (Figure 3, columns c and d). Two to three fold increases in NF-kB concentration were observed after TNF-alpha stimulation of PC3 cells for 15 minutes.

Key Advantages

• Quantitative measurement of NF-kB using a standard curve.
• Protocols available for measurement of either activated/nuclear NF-kB, or total NF-kB present in the cell/biological sample.
• Greater sensitivity than gel-shift assay or ELISA with absorption detection.
• Shorter time than gel shift – assay may be completed in 3-5 h post sample preparation
• No radioactive chemicals used.
• Compatible with high-throughput applications: 96-well format allows simultaneous measurement of up to 40 samples in duplicate along with the standard curve.