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HTRF Human and Mouse Total RNA Pol II Detection Kit, 10,000 Assay Points

Picture RNA pol 2 total kit
HTRF Human and Mouse Total RNA Pol II Detection Kit, 10,000 Assay Points
Picture RNA pol 2 total kit
Picture RNA pol 2 total kit
Picture RNA pol 2 total kit
Picture RNA pol 2 total kit
Picture RNA pol 2 total kit

This HTRF kit enables the cell-based quantitative detection of Total RNA Pol II

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Feature Specification
Application Protein Quantification
Sample Volume 16 µL
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This HTRF kit enables the cell-based quantitative detection of Total RNA Pol II

View product information
Click to copy promo code to clipboard.
img-icon-10-off-white-yellow.svg

SAVE 10% on your first Revvity.com order. Use promo code below.

HELLO10

Terms and conditions apply.

Product Variants
Unit Size: 500 assay points
Part #:
64RNAP2TPEG
List Price
USD 2,147.00
Your online price:
Unit Size: 10,000 assay points
Part #:
64RNAP2TPEH
List Price
USD 12,490.00
Your online price:
For research use only. Not for use in diagnostic procedures. All products to be used in accordance with applicable laws and regulations including without limitation, consumption, and disposal requirements under European REACH regulations (EC 1907/2006).
Total list price:
USD 12,490.00
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Picture RNA pol 2 total kit
Picture RNA pol 2 total kit
HTRF Human and Mouse Total RNA Pol II Detection Kit, 10,000 Assay Points
Picture RNA pol 2 total kit
Picture RNA pol 2 total kit
Picture RNA pol 2 total kit
HTRF Human and Mouse Total RNA Pol II Detection Kit, 10,000 Assay Points
Picture RNA pol 2 total kit
Picture RNA pol 2 total kit

Product information

Overview

RNA polymerase II (RNA Pol II) is one of the primary enzymes responsible for expressing protein-encoding genes and some small nuclear RNAs. The carboxy-terminal domain (CTD) of RNA Pol II undergoes a cycle of phosphorylation, catalyzed mainly by members of the cyclin-dependent kinase (CDK) family. RNA Pol II CTD phosphorylation allows it to temporally couple transcription with transcription-associated processes (binding specificity of the CTD for particular factors is determined by the site-specific phosphorylation/dephosphorylation events).

Specifications

Application
Protein Quantification
Brand
HTRF
Buffer/Solvent
Lysis Buffer 1
Detection Modality
HTRF
Host Species
Human
Mouse
Molecular Modification
Total
Product Group
Kit
Sample Volume
16 µL
Shipping Conditions
Shipped in Dry Ice
Target
RNA pol II
Target Class
Phosphoproteins
Target Species
Human
Mouse
Technology
TR-FRET
Therapeutic Area
Immuno-oncology
Inflammation
Unit Size
10,000 assay points

How it works

Total RNA Pol II assay principle

The Total RNA Pol II assay quantifies the expression level of RNA Pol II in a cell lysate. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The Total RNA Pol II assay uses two labeled antibodies, one coupled to a donor fluorophore and the other to an acceptor. Both antibodies are highly specific for a distinct epitope on the protein. In the presence of RNA Pol II in a cell extract, the addition of these conjugates brings the donor fluorophore into close proximity with the acceptor and thereby generates a FRET signal. Its intensity is directly proportional to the concentration of the protein present in the sample, and provides a means of assessing the protein's expression under a no-wash assay format.

R&D-HTRF-attributes assay principle total (with phospho)
Total RNA Pol II two-plate assay protocol

The two-plate protocol involves culturing cells in a 96-well plate before lysis, then transferring lysates into a 384-well low volume detection plate before the addition of HTRF Total RNA Pol II detection reagents. This protocol enables the cells' viability and confluence to be monitored.

2-plates assay protocol (how it works)
Total RNA Pol II one-plate assay protocol

Detection of Total RNA Pol II with HTRF reagents can be performed in a single plate used for culturing, stimulation, and lysis. No washing steps are required. This HTS designed protocol enables miniaturization while maintaining robust HTRF quality.

1-plate assay protocol

Assay validation

Inhibition of Phospho-Ser2 and Ser5 RNA Pol II with Flavopiridol in HeLa cells

HeLa cells were plated in a 96-well plate (40,000 cells/well) in complete culture medium, and incubated overnight at 37°C, 5% CO2. Then, cells were treated with a dose response of Flavopiridol, a CDK9 inhibitor, for 2 h. After culture medium removal, cells were lysed with 50 µL of supplemented Lysis Buffer #1 for 30 min at room temperature under gentle shaking. After cell lysis, 16 µL of cell lysate were transferred into a HTRF 384-well low volume white microplate then 4 µL of the Total RNA Pol II or HTRF Phospho-RNA Pol II (Ser2) (Revvity, #64RNAP2S2PEG/PEH/PEY) or HTRF Phospho-RNA Pol II (Ser5) (Revvity, #64RNAP2S5PEG/PEH/PEY) detection reagents were added. HTRF signal for three kits was recorded using Envision Nexus reader after an overnight-incubation at room temperature. In parallel, cell viability was assessed. To do this, 5 µL of the same cell lysate were transferred into an HTRF 96-well low volume white plate, and 25 µL of ATPlite 1step detection reagent were added (Revvity®, #6016736/1/9). The luminescence signal was measured after a 10-min incubation in the dark at room temperature.

As expected, Flavopiridol, a CDK9 inhibitor, inhibited RNA Pol II phosphorylation on Serines 2 and 5, leading to a dose-dependent decrease of Phospho-RNA Pol II (Ser2) and Phospho-RNA Pol II (Ser5) signals, without any significant effect on the expression level of the total RNA Pol II protein. In addition, this treatment did not induce cytotoxic effects, as measured by the cell viability indicator ATPLite (data not shown).

assay validation inhibitor Flavopiridol RNA Pol II total (64RNAP2TPEG, 64RNAP2TPEH

 

Inhibition of Phospho-Ser2 and -Ser5 RNA Pol II with Thal-SNS-032 in HEK293 cells

HEK293 cells were plated in a 96-well plate (50,000 cells/well) in complete culture medium, and incubated overnight at 37°C, 5% CO2. Then, cells were treated with a dose response of Thal-SNS-032, a selective CDK9 degrader, for 4 h. After culture medium removal, cells were lysed with 50 µL of supplemented Lysis Buffer #1 for 30 min at room temperature under gentle shaking. After cell lysis, 16 µL of cell lysate were transferred into a HTRF 384-well low volume white microplate then 4 µL of the Total RNA Pol II or HTRF Phospho-RNA Pol II (Ser2) (Revvity®, #64RNAP2S2PEG/PEH/PEY) or HTRF Phospho-RNA Pol II (Ser5) (Revvity®, #64RNAP2S5PEG/PEH/PEY) or Total CDK9 (Revvity®, #64CDK9TPEG/PEH/PEY) detection reagents were added. HTRF signal for four kits was recorded using HTRF compatible reader after an overnight-incubation at room temperature. In parallel, cell viability was assessed. To do this, 5 µL of the same cell lysate were transferred into an HTRF 96-well low volume white plate, and 25 µL of ATPlite 1step detection reagent were added (Revvity®, #6016736/1/9). The luminescence signal was measured after a 10-min incubation in the dark at room temperature.

As expected, Thal-SNS-032, a selective CDK9 degrader, inhibited RNA Pol II phosphorylation on Serines 2 and 5 and decreased CDK9 expression level, without any significant effect on the expression level of the total RNA Pol II protein. In addition, this treatment did not induce cytotoxic effects, as measured by the cell viability indicator ATPLite (data not shown).

assay validation inhibitor Thal SNS 032 RNAPol2 total

 

Inhibition of Phospho-Ser5 RNA Pol II with BI-1347 in HEK293 cells

HEK293 cells were plated in a 96-well plate (50,000 cells/well) in complete culture medium, and incubated overnight at 37°C, 5% CO2. Then, cells were treated with a dose response of BI-1347, a CDK8/cyclin C inhibitor, for 4 h. After culture medium removal, cells were lysed with 50 µL of supplemented Lysis Buffer #1 for 30 min at room temperature under gentle shaking. After cell lysis, 16 µL of cell lysate (neat for Total RNA Pol II and Phospho-RNA Pol II (Ser2) assays and dilution 1/4 for Phospho-RNA Pol II (Ser5) assay) were transferred into a HTRF 384-well low volume white microplate then 4 µL of the Total RNA Pol II or HTRF Phospho-RNA Pol II (Ser2) (Revvity®, #64RNAP2S2PEG/PEH/PEY) or HTRF Phospho-RNA Pol II (Ser5) (Revvity®, #64RNAP2S5PEG/PEH/PEY) detection reagents were added. HTRF signal for three kits was recorded using an HTRF compatible reader after an overnight-incubation at room temperature. In parallel, cell viability was assessed. To do this, 5 µL of the same cell lysate were transferred into an HTRF 96-well low volume white plate, and 25 µL of ATPlite 1step detection reagent were added (Revvity®, #6016736/1/9). The luminescence signal was measured after a 10-min incubation in the dark at room temperature.

As expected, BI-1347, a CDK8/cyclin C inhibitor, inhibited RNA Pol II phosphorylation on Serine 5, leading to a dose-dependent decrease of Phospho-RNA Pol II (Ser5) signal, without any significant effect on the expression level of the total RNA Pol II protein and phosphorylation level on Serine 2. In addition, this treatment did not induce cytotoxic effects, as measured by the cell viability indicator ATPLite (data not shown).

assay validation inhibitor BI 1347 RNA Pol II total
Selectivity of Total RNA Pol II assay using phospho-peptides competition

HeLa cells were cultured in flasks (4 millions / 175 cm2) in complete culture medium, and incubated 48 h at 37°C, 5% CO2. After culture medium removal, cells were lysed with 3 mL of supplemented Lysis Buffer #1 for 30 min at room temperature under gentle shaking. After cell lysis, 14 µL of cell lysate were transferred into a HTRF 384-well low volume white microplate then 2 µL of peptide (Non-phosphorylated or Phospho-Ser2 or Phospho-Ser5 or Phospho-Ser7 or Phospho-Ser2-Ser5-Ser7, named Triple phospho) and 4 µL of the HTRF Total RNA Pol II detection reagents were added. HTRF signal was recorded using an HTRF compatible reader after an overnight-incubation at room temperature.

As expected, none peptide had an impact on Total RNA Pol II signal, demonstrating that RNA Pol II phosphorylations didn’t interfere with the detection of Total RNA Pol II protein.

assay validation specificity RNA Pol II total (64RNAP2TPEG, 64RNAP2TPEH, 64RNAP2TPEY)
Assessment of Total RNA Pol II level in various human cell lines

HEK293, MCF7 and A431 cells were cultured in flasks in complete culture medium, and incubated 48 h at 37°C, 5% CO2. After culture medium removal, cells were lysed with 3 mL of supplemented Lysis Buffer #1 for 30 min at room temperature under gentle shaking. After cell lysis, 16 µL of cell lysate (dilution 1/3) were transferred into a HTRF 384-well low volume white microplate then 4 µL of the HTRF Total RNA Pol II detection reagents were added. HTRF signal was recorded using HTRF compatible reader after an overnight-incubation at room temperature.

The HTRF Total RNA Pol II assay efficiently detected Total RNA Pol II in various cellular models expressing different levels of the protein.

assay validation versatility RNA Pol II total (64RNAP2TPEG, 64RNAP2TPEH, 64RNAP2TPEY)
HTRF Total RNA Pol II assay compared to Western Blot

HEK293 cells were cultured in flasks and incubated 48 h at 37°C, 5% CO2. After culture medium removal, cells were lysed with 3 mL of supplemented Lysis Buffer #1 for 30 min at room temperature under gentle shaking. Equal amounts of cell lysates (16 µL) were used for a side-by-side comparison of Western Blot and HTRF techniques.

Using the HTRF Total RNA Pol II assay, 780 cells/well were enough to detect a significant signal, while more than 12,500 cells were needed to obtain a minimal chemiluminescent signal using Western Blot. Therefore, in these conditions, the HTRF Total RNA Pol II assay is 16 times more sensitive than Western Blot technique.

assay validation WB RNA Pol II total (64RNAP2TPEG, 64RNAP2TPEH, 64RNAP2TPEY)

Simplified pathway

RNA Polymerase II signaling pathway

RNA Polymerase is under the regulation of CDKs such as CDK7 and CDK9, which are involved and expressed at different stage of the cellular cycle. RNA Polymerase’s role is to perform the transcription of DNA into mRNA, which it does by binding DNA promoter sequences and recruiting several partners to form a trancription complex capable of initiating the separation of the two DNA strands, RNA Pol II then moves along the strand and pieces together a matching mRNA strand from available nucleotides.

The activity of RNA Pol II is regulated by the phosphorylation state of its tail which can get sequentially phosphorylated at Ser7, Ser5 and Ser2. When unphosphorylated, the polymerase remains inactive, but when Ser7 and Ser5 become phosphorylatated they enable the assembly of the transcription complex. This is followed by the phosphorylation of Ser2 and RNA Pol II becoming fully active and mobile along the open DNA strand. As it performs transcription, RNA Pol II is progressively dephosphorylated at Ser5 then Ser7, and returns to an inactive state that detaches from the DNA strand,

The cycle of phosphorylation and desphosphorylation ensures the activation of RNA Pol II remains temporary and under control.

Simplified pathway RNA pol II total (64RNAP2TPEG, 64RNAP2TPEH, 64RNAP2TPEY)

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