Chromatin immunoprecipitation analysis of SPT16 was performed using cross-linked chromatin from 1 x 106 HCT116 colon carcinoma cells treated with serum for 0, 15, 30, and 60 minutes. Immunoprecipitation was performed using a multiplex microplate Matrix ChIP assay (see reference for Matrix ChIP protocol: http://www.ncbi.nlm.nih.gov/pubmed/22098709) with 1.0ul/100ul well volume of an SPT16 polyclonal antibody (Product # PA1-12697). Chromatin aliquots from ~1 x 105 cells were used per ChIP pull-down. Quantitative PCR data were done in quadruplicate using 1ul of eluted DNA in 2ul SYBR real-time PCR reactions containing primers to amplify -15kb upstream of the Egr1 gene or exon-1 of Egr1. PCR calibration curves were generated for each primer pair from a dilution series of sheared total genomic DNA. Quantitation of immunoprecipitated chromatin is presented as signal relative to the total amount of input chromatin. Results represent the mean +/- SEM for three experiments. A schematic representation of the Egr-1 locus is shown above the data where boxes represent exons (black boxes = translated regions, white boxes = untranslated regions); the zigzag line represents an intron; and the straight line represents upstream sequence. Regions amplified by Egr-1 primers are represented by black bars. Data courtesy of the Innovators Program.
|Tested species reactivity||Human, Mouse|
|Host / Isotype||Rabbit / IgG|
|Immunogen||Recombinant protein corresponding to residues 321-640 of human SPT16, conjugated to KLH.|
|Purification||Ammonium sulfate precipitation|
|Contains||0.08% sodium azide|
|Storage Conditions||-20° C, Avoid Freeze/Thaw Cycles|
|Tested Applications||Dilution *|
|ChIP assay (ChIP)||1-3 ul|
|Immunoprecipitation (IP)||Assay Dependent|
|Western Blot (WB)||5-10 µg/ml|
* 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.
SPT16 and SSRP1 are found in all eurkaryotic cells and exists as part of an essential heterodimer complex. Together, SPT16 and SSRP1 are a highly abundant nuclear complex that in mammals has been called FACT, or Facilitates Chromatin Transcription. FACT enhances the processivity of RNA polymerase II through nucleosomes. Evidence also points to a role for FACT in replication, possibly by enhancing DNA polymerase through nucleosomes as well.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.