AERIUS® IMAGER
Applications for the Aerius Imaging System
TRANSCRIPTION FACTOR ASSAY
Application Overview
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 System by substituting IRDye® secondary antibody for HRP conjugate1.
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
[ABOVE] 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.
[ABOVE] 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).
1 Cayman Chemical PPARγ Transcription Factor Kit Assay on the Odyssey System, LI-COR Biosciences (2007)
2 Active Motif TransAM™ Transcription Factor Assays LI-COR Biosciences (2006)
Technical Resources
Cayman Chemical PPARγ Transcription Factor Kit Assay on the Odyssey System,
LI-COR Biosciences (2007)Active Motif TransAM ™ Transcription Factor Assays
LI-COR Biosciences (2006)
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