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Synthetic peptide of Saccharomyces cerevisiae RNA polymerase II CTD repeat YSPTSPS, phosphorylated at S5.
(Peptide available as ab18488.)
Phosphorylation of RNA polymerase II's largest subunit C-terminal domain (CTD) is a key event during mRNA metabolism.
Our Abpromise guarantee covers the use of ab5131 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|IHC-P||Use at an assay dependent concentration. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.|
|IHC - Wholemount||Use at an assay dependent concentration.|
|IHC-Fr||Use at an assay dependent concentration.|
|CHIPseq||Use at an assay dependent concentration.|
|WB||1/1000. Detects a band of approximately 240 kDa (predicted molecular weight: 217 kDa).Can be blocked with Human RNA polymerase II CTD repeat YSPTSPS (phospho S5) peptide (ab18488).|
|ChIP||Use 2-25 µg for µg of chromatin.|
|ICC/IF||Use a concentration of 1 µg/ml.|
Chromatin was prepared from U2OS cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10 mins. The ChIP was performed with 25 µg of chromatin, 2 µg of ab5131 (blue), and 20 µl of Protein A/G sepharose beads. No antibody was added to the beads control (yellow). The immunoprecipitated DNA was quantified on the inactive AFM and F8 promoters, the GAPDH promoter (active) and over the y-Actin gene (active). Schematic diagram of the y-Actin gene is shown on the top of the figure. Black boxes represent exons and thin lines represent introns. PCR products are depicted as bars under the gene.
Western blot using ab5131.
Lane 1: ab5131 at 1/500
Lane 2: ab5131 at 1/2000
Lane 3: ab5131 at 1/500 blocked with phospho peptide
Lane 4: ab5131 at 1/500 blocked with non-phospho control peptide
Phospho peptide is YSPTSpPSYSPTSpPS-GGC (ab18488)
Non-phospho control peptide is YSPTSPSYSPTSPS-GGC (ab12795)
Secondary ab: Goat anti-rabbit HRP (IgG) ab6721 (1/5000)
Lanes 1 to 4: 20µg of HeLa nuclear extract per lane
Blocking peptides used at 1µg/ml.
Exposure time: 30 seconds
Expected molecular weight: ~240 kDa
This image is courtesy of an anonymous Abreview
IHC - Wholemount of Caenorhabditis elegans larvae labelling RNA polymerase II CTD repeat YSPTSPS (phospho S5) with ab5131. The sample was incubated with primary antibody (1/500 in PBS + 3% BSA + 0.1% Triton X-100) for 12 hours at 4°C. ab150077, a goat anti-rabbit Alexa 488 - (1/1000), was used as the secondary antibody.