ChIP assays were performed using human osteosarcoma (U-2 OS) cells, the anti-TBP antibody (Cat. no. 49-1036), and optimized PCR primer sets (c-fos promoter (Cat. no 49-2029), myoglobin exon 2, GAPDH promoter, GAPDH promoter (0.5 kb), and GAPDH promoter (1.0 kb) for qPCR. Each ChIP experiment used sheared chromatin from 1 million cells and 4 µg of anti-TBP antibody. Recovery (% ChIP⁄input) is shown in the top graph. Occupancy (fold: +ve⁄-ve) is in the bottom graph. Recovery and occupancy of the c-fos promoter by TBP (red bars); recovery and occupancy of the GAPDH promoter by TBP (green bars) are shown. Occupancy of the two promoters by TBP is evident based on fluorescent qPCR analysis of immunoprecipitated DNA. Controls for IP and PCR specificity include primers for the myoglobin exon 2 (black) as well as GAPDH (0.5 kb, pink) and GAPDH (1 kb, blue).
|Tested species reactivity||Human|
|Host / Isotype||Mouse / IgG1|
|Immunogen||Raised against the amino-terminal domain of human TBP.|
|Storage Conditions||-20° C, Avoid Freeze/Thaw Cycles|
|Tested Applications||Dilution *|
|ChIP assay (ChIP)||0.5 ul|
* Suggested working dilutions are given as a guide only. It is recommended that the user titrate the product for use in their own experiment using appropriate negative and positive controls.
Initiation of transcription by RNA polymerase II requires the activities of more than 70 polypeptides. The protein that coordinates these activities is transcription factor IID (TFIID), which binds to the core promoter to position the polymerase properly, serves as the scaffold for assembly of the remainder of the transcription complex, and acts as a channel for regulatory signals. TFIID is composed of the TATA-binding protein (TBP) and a group of evolutionarily conserved proteins known as TBP-associated factors or TAFs. TAFs may participate in basal transcription, serve as coactivators, function in promoter recognition or modify general transcription factors (GTFs) to facilitate complex assembly and transcription initiation. A distinctive feature of TBP is a long string of glutamines in the N-terminal. This region of the protein modulates the DNA binding activity of the C terminus, and modulation of DNA binding affects the rate of transcription complex formation and initiation of transcription. Mutations that expand the number of CAG repeats encoding this polyglutamine tract, and thus increase the length of the polyglutamine string, are associated with spinocerebellar ataxia 17, a neurodegenerative disorder classified as a polyglutamine disease.
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