NFkB Filter Plate Assay

Nuclear factor-kB (NFkB) is a transcription factor that controls the expression of genes involved in immune responses, apoptosis, and cell cycle. NFkB can be activated by a variety of stimuli, including cytokines, T and B cell mitogens, viral proteins, and stress inducers. The stimulation leads to phosphorylation of its inhibitor IkB, which releases NF-kB from IkB complex. After dissociation, NFkB translocates from the cytoplasm into the nucleus, where it binds to its target DNA elements and positively regulates the transcription of its target genes. Monitoring the activity of NFkB is crucial to the studies of the mechanism underlying NFkB activation and its associated pathway. Signosis has developed NFkB filter assay. Unlike conventional gel shift assays, NFkB filter assay is a high throughput assay and it can analyze multiple samples simultaneously. Meanwhile, the assay can be set up for a few samples with less tedious than non-radioactive gel shift assay or much safer than 32P-based gel shift assay.

A. Benefits:

Signosis proprietary NFkB filter assay offers following benefits:

  • Simple and quick -It is simpler and quicker than gel shift assay.

  • High throughput screen- Multiple samples can be analyzed in one single assay.

  • Cost-effective detection- The cost is lower than ELISA-based TF assays.

  • Quantitative comparison The difference of two or more samples in TF activity can be quantitatively analyzed.

B. Principle of the technology

Signosis’ NFkB filter assay is a plate-based analysis for monitoring the activity of NFkB. In the assay, two plates – filter plate and hybridization plate – are needed. In the assay, pre-labeled DNA binding sequence of NFkB is mixed with nuclear extract to allow formation of NFkB and the pre-labeled DNA sequence. The filter is used to retain bound DNA sequence and remove free DNA sequence. The bound pre-labeled DNA sequence is then eluted from the filter and collected for plate hybridization. The captured DNA sequence is further detected with streptavidin-HRP. Luminescence is reported as relative light units (RLUs) on a microplate luminometer.

Figure 1
Diagram

Figure2. NFkB filter assay. HeLa cells were treated with or without TNF. Nuclear extracts were prepared and subjected to tNFkB filter assay. The bound NFkB probe was measured with Luminescence.

 

 Product Size Cat. Price
NFkB Filter Plate Assay 96 FA-0001 $419
  • NFkB Filter Plate Assay User Manual (PDF)

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