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Anti-RNA polymerase II subunit B1 (phospho-CTD Ser-5) Antibody, clone 3E8 is a highly specific rat monoclonal antibody, that targets RNA Polymerase & has been tested in western blotting & ELISA.
More>>Anti-RNA polymerase II subunit B1 (phospho-CTD Ser-5) Antibody, clone 3E8 is a highly specific rat monoclonal antibody, that targets RNA Polymerase & has been tested in western blotting & ELISA. Less<<
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Anti-RNA polymerase II subunit B1 (phospho-CTD Ser-5) Antibody, clone 3E8
Alternate Names
DNA-directed RNA polymerase II subunit RPB1
RNA polymerase II subunit B1
DNA-directed RNA polymerase II subunit A
DNA-directed RNA polymerase III largest subunit
RNA-directed RNA polymerase II subunit RPB1
Background Information
RPB1 (RNA polymerase II subunit B1) is the catalytic component of RNA polymerase II which synthesizes mRNA and non-coding RNAs. During transcription, elongation is influenced by the phosphorylation status of the C-terminal domain (CTD) of RPB1, which acts as an assembly platform for factors regulating transcription initiation, elongation, termination and mRNA processing. Phosphorylation occurs mainly at residues 'Ser-2' and 'Ser-5' of the tandem 7 residue repeats in the C-terminal domain (CTD), which activates Pol II. This phosphorylation also can occur at 'Ser-7' of the heptapepdtide repeat. This antibody recognizes the 'Ser-5' CTD residue of RPB1.
References
Product Information
Format
Culture Supernatant
Control
Untreated and lambda phosphotase treated NIH/3T3 cell lysates
Anti-RNA polymerase II subunit B1 (phospho-CTD Ser-5) Antibody, clone 3E8 is a highly specific rat monoclonal antibody, that targets RNA Polymerase & has been tested in western blotting & ELISA.
Key Applications
Chromatin Immunoprecipitation (ChIP)
Western Blotting
ELISA
Application Notes
Chromatin Immunoprecipitation Analysis: A representative lot was used by an independent laboratory to immunoprecipitate RNA polymerase II subunit B1 (phospho-CTD Ser-5) in ChIP (Chapman, R., et al. (2007). Science. 318(5857):1780 -1782.).
ELISA Analysis: A representative lot was used by an independent laboratory to detect RNA polymerase II subunit B1 (phospho-CTD Ser-5) in ChIP (Chapman, R., et al. (2007). Science. 318(5857):1780 -1782.).
Biological Information
Immunogen
Ovalbumin-conjugated linear peptide corresponding to human RNA Polymerase II subunit B1 phosphorylated at Ser5 of the C-terminal domain (CTD).
Epitope
Phosphorylated Ser5 at the C-terminus domain (CTD).
Clone
3E8
Host
Rat
Specificity
This antibody recognizes RNA Polymerase II subunit B1 phosphorylated at Ser5 of the C-terminus domain (CTD).
Isotype
IgG2aκ
Species Reactivity
Mouse
Species Reactivity Note
Demonstrated to react with Mouse. Predicted to react with Human based upon immunogen design.
Evaluated by Western Blot in untreated and lambda phosphotase treated NIH/3T3 cell lysates.
Western Blot Analysis: A 1:2,000 dilution from a representative lot detected RNA polymerase II subunit B1 (phospho-CTD Ser-5) in 10 µg of untreated and lambda phosphotase treated NIH/3T3 cell lysates.
Usage Statement
Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.
Storage and Shipping Information
Storage Conditions
Stable for 1 year at -20°C from date of receipt. Handling Recommendations: Upon receipt and prior to removing the cap, centrifuge the vial and gently mix the solution. Aliquot into microcentrifuge tubes and store at -20°C. Avoid repeated freeze/thaw cycles, which may damage IgG and affect product performance.
Transcription steps are marked by different modifications of the C-terminal domain of RNA polymerase II (RNAPII). Phosphorylation of Ser5 and Ser7 by cyclin-dependent kinase 7 (CDK7) as part of TFIIH marks initiation, whereas phosphorylation of Ser2 by CDK9 marks elongation. These processes are thought to take place in localized transcription foci in the nucleus, known as "transcription factories," but it has been argued that the observed clusters/foci are mere fixation or labeling artifacts. We show that transcription factories exist in living cells as distinct foci by live-imaging fluorescently labeled CDK9, a kinase known to associate with active RNAPII. These foci were observed in different cell types derived from CDK9-mCherry knock-in mice. We show that these foci are very stable while highly dynamic in exchanging CDK9. Chromatin immunoprecipitation (ChIP) coupled with deep sequencing (ChIP-seq) data show that the genome-wide binding sites of CDK9 and initiating RNAPII overlap on transcribed genes. Immunostaining shows that CDK9-mCherry foci colocalize with RNAPII-Ser5P, much less with RNAPII-Ser2P, and not with CDK12 (a kinase reported to be involved in the Ser2 phosphorylation) or with splicing factor SC35. In conclusion, transcription factories exist in living cells, and initiation and elongation of transcripts takes place in different nuclear compartments.
RNA polymerase II is distinguished by its large carboxyl-terminal repeat domain (CTD), composed of repeats of the consensus heptapeptide Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7. Differential phosphorylation of serine-2 and serine-5 at the 5' and 3' regions of genes appears to coordinate the localization of transcription and RNA processing factors to the elongating polymerase complex. Using monoclonal antibodies, we reveal serine-7 phosphorylation on transcribed genes. This position does not appear to be phosphorylated in CTDs of less than 20 consensus repeats. The position of repeats where serine-7 is substituted influenced the appearance of distinct phosphorylated forms, suggesting functional differences between CTD regions. Our results indicate that restriction of serine-7 epitopes to the Linker-proximal region limits CTD phosphorylation patterns and is a requirement for optimal gene expression.
