To characterize transcription factors (TFs) that binds to a specific promoter or that regulate the expression of a specific gene via its upstream promoter, two common approaches are applied. First is to employ gel shift assay with DNA binding sites of TFs that are silico-identified within the promoter. Second is to removal or knockout the binding site(s) of a specific TF in order to measure whether the expression of a promoter-linked reporter is increased or decreased. Often, a series of reporter constructs with the promoter deletions or mutations need to make because many binding sites of one or a few TFs are present within a promoter. Signosis has developed a fast method to facilitate the characterization of promoters through a revised TF activation array. This assay will help to test whether a selected 96 TFs bind to the promoter or not.
List of Applicable TFs:
Promoter-binding TF profiling plate arrays are a competition assay of Signosis’ TF activation plate arrays. In the TF activation plate arrays, if all of 48 or 96 targeted transcription factors exist in the assayed samples, they will form 48 or 96 types of complexes, each TF with its corresponding biotin-labeled oligo (just like the complex in the gel shift assay). After a simple spin separation of the complexes from unbound free biotin-labeled oligos with a membrane-based column, TF-bound oligos eluted from the column and used for plate hybridization in which a complementary DNA of biotin-labeled oligo. The captured oligo is then detected with streptavidin-HRP and a chemiluminescent substrate. If any TF is not present, it will not form a complex, leading to no detection of TF in the following plate assay of biotin-labeled oligo. In promoter-binding TF profiling arrays, PCR fragment containing the promoter of your interest is mixed with a set of 48 or 96 biotin-labeled oligos corresponding to 48 or 96 TFs along with an assayed sample. If unlabeled promoter DNA fragment contains a TF binding sequence, it will compete with the biotin-labeled oligo to bind to the TF in the sample, leading to no or less biotin labeled TF/DNA complex formation and no or lower detection. Through comparison in the presence and absence of the competitor promoter DNA fragment, promoter-bound TFs can be identified.
Promoter-Binding TF Profiling Assay: HeLa cells were treated with or without PMA. PMA was used to acticve TFs including AP1 and NFkB. Nuclear extracts were prepared and incubated with TF binding oligo probe mix: control HeLa cells without PMA treatment with the probe mix (blue); PMA-treated HeLa cells with the probe mix alone (red) and the probe mix plus IL6 promoter DNA fragment (yellow).
LiteratureView user manual
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