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Transcription Factor Assay

PPARy Assay

Figure 1. PPARγ Assay (Cayman Chemical). A.) Comparison of the data using increasing amounts (0, 5, 10, 15, and 20 μL) of positive control lysate, with either the HRP antibody or IRDye 800CW antibody (Odyssey) for detection. B.) Comparison of reduction in signal when increasing amounts of competitor DNA are added. Note that the increased sensitivity of the assay using IRDye 800CW results in a more gradual drop-off in the signal with higher amounts of competitor, while the HRP detection signal drops sharply with the addition of either 5 or 10 μl of competitor DNA.

ELISA-format assays can be a convenient alternative to gel mobility shift assays (EMSA) for analysis of transcription factor binding.

  • Cayman Chemical (Ann Arbor, MI) and ActiveMotif (Carlsbad, CA) Transcription Factor Assays use a 96-well format to perform nonradioactive detection of DNA binding activity in nuclear extracts.
  • 8-well strips are pre-coated with double-stranded DNA containing the consensus binding site for the transcription factor.
  • Target proteins in nuclear extracts bind to the consensus site and are detected with primary and secondary antibodies.
  • Competitor DNA is used to confirm assay specificity.

These kits use an HRP conjugate for detection, but are easily converted for detection with the Odyssey® CLx or Odyssey Sa System by substituting IRDye® secondary antibody for HRP conjugate.1

  • Reduces assay time and eliminates need to stop the reaction
  • Allows dry assay plates to be stored in the dark and scanned at your convenience
p53 TransAM Assay

Figure 2. p53 TransAM Assay (ActiveMotif). HRP secondary antibody was replaced with IRDye 800CW secondary antibody. A.) Odyssey image of the assay strips. Row 1 shows Raji nuclear extracts (15, 12.5, 10, 7.5, 5, 2.5, 1.25, and 0 μg) with free wild type consensus oligonucleotide. Row 2 shows Raji nuclear extracts (same amounts) without competitor oligonucleotide. B.) Quantification of the assay (negative control was subtracted as background).

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