POLR2A (Phospho-Ser1619) Colorimetric Cell-Based ELISA Kit

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SKU:
CBCAB01248
Product Type:
ELISA Kit
ELISA Type:
Cell Based Phospho Specific
Research Area:
Immunology
Reactivity:
Human
Mouse
Rat
Detection Method:
Colorimetric
Frequently bought together:

Description

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POLR2A (Phospho-Ser1619)Colorimetric Cell-Based ELISA Kit

The Human POLR2A (RNA polymerase II subunit A) Phospho-Ser1619 Colorimetric Cell-based ELISA Kit is specifically designed for the accurate and sensitive detection of phosphorylated POLR2A at serine 1619 in cell lysates. This kit offers high specificity and sensitivity, allowing for reliable and reproducible results in a variety of research applications.POLR2A is a key component of RNA polymerase II, playing a critical role in transcription. Phosphorylation at serine 1619 of POLR2A is known to regulate transcriptional activity and gene expression.

Dysregulation of POLR2A phosphorylation has been linked to various diseases, including cancer and neurologic disorders, making it a valuable biomarker for studying these conditions and potential therapeutic interventions.With its advanced technology and exceptional performance, the Human POLR2A Phospho-Ser1619 Colorimetric Cell-based ELISA Kit is an essential tool for researchers studying transcriptional regulation, gene expression, and the role of POLR2A phosphorylation in disease development and progression.

Product Name:POLR2A (Phospho-Ser1619) Colorimetric Cell-Based ELISA
Product Code:CBCAB01248
ELISA Type:Cell-Based
Target:POLR2A (Phospho-Ser1619)
Reactivity:Human, Mouse, Rat
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nm
Format:2 x 96-Well Microplates

The POLR2A (Phospho-Ser1619) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect POLR2A protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated POLR2A in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on POLR2A phosphorylation.

Qualitative determination of POLR2A (Phospho-Ser1619) concentration is achieved by an indirect ELISA format. In essence, POLR2A (Phospho-Ser1619) is captured by POLR2A (Phospho-Ser1619)-specific primary (1ø) antibodies while the HRP-conjugated secondary (2ø) antibodies bind the Fc region of the 1ø antibody. Through this binding, the HRP enzyme conjugated to the 2ø antibody can catalyze a colorimetric reaction upon substrate addition. Due to the qualitative nature of the Cell-Based ELISA, multiple normalization methods are needed:

1. A monoclonal antibody specific for human GAPDH is included to serve as an internal positive control in normalizing the target absorbance values.
2. Following the colorimetric measurement of HRP activity via substrate addition, the Crystal Violet whole-cell staining method may be used to determine cell density. After staining, the results can be analysed by normalizing the absorbance values to cell amounts, by which the plating difference can be adjusted.
Database Information:Gene ID: 5430, UniProt ID: P24928, OMIM: 180660, Unigene: Hs.270017
Gene Symbol:POLR2A
Sub Type:Phospho
UniProt Protein Function:POLR2A: DNA-dependent RNA polymerase catalyzes the transcription of DNA into RNA using the four ribonucleoside triphosphates as substrates. Largest and catalytic component of RNA polymerase II which synthesizes mRNA precursors and many functional non-coding RNAs. Forms the polymerase active center together with the second largest subunit. Pol II is the central component of the basal RNA polymerase II transcription machinery. It is composed of mobile elements that move relative to each other. RPB1 is part of the core element with the central large cleft, the clamp element that moves to open and close the cleft and the jaws that are thought to grab the incoming DNA template. At the start of transcription, a single stranded DNA template strand of the promoter is positioned within the central active site cleft of Pol II. A bridging helix emanates from RPB1 and crosses the cleft near the catalytic site and is thought to promote translocation of Pol II by acting as a ratchet that moves the RNA-DNA hybrid through the active site by switching from straight to bent conformations at each step of nucleotide addition. During transcription elongation, Pol II moves on the template as the transcript elongates. Elongation is influenced by the phosphorylation status of the C-terminal domain (CTD) of Pol II largest subunit (RPB1), which serves as a platform for assembly of factors that regulate transcription initiation, elongation, termination and mRNA processing. Acts as a RNA- dependent RNA polymerase when associated with small delta antigen of Hepatitis delta virus, acting both as a replicate and transcriptase for the viral RNA circular genome. Component of the RNA polymerase II (Pol II) complex consisting of 12 subunits. The phosphorylated C-terminal domain interacts with FNBP3 and SYNCRIP. Interacts with SAFB/SAFB1. Interacts with CCNL1 and MYO1C. Interacts with CCNL2 and SFRS19. Component of a complex which is at least composed of HTATSF1/Tat-SF1, the P-TEFb complex components CDK9 and CCNT1, RNA polymerase II, SUPT5H, and NCL/nucleolin. Interacts with PAF1. Interacts (via C-terminus) with FTSJD2, CTDSP1 and SCAF8. Interacts via the phosphorylated C-terminal domain with WDR82 and with SETD1A and SETD1B only in the presence of WDR82. Interacts with ATF7IP. When phosphorylated at 'Ser-5', interacts with MEN1; the unphosphorylated form, or phosphorylated at 'Ser-2' does not interact. Interacts with DDX5. Belongs to the RNA polymerase beta' chain family.
UniProt Protein Details:

Protein type:Transferase; EC 2.7.7.6; Nucleotide Metabolism - purine; Transcription initiation complex; EC 2.7.7.48; Nucleotide Metabolism - pyrimidine

Chromosomal Location of Human Ortholog: 17p13.1

Cellular Component: DNA-directed RNA polymerase II, core complex; nucleolus; nucleoplasm; nucleus

Molecular Function:DNA binding; DNA-directed RNA polymerase activity; metal ion binding; protein binding; RNA-directed RNA polymerase activity; ubiquitin protein ligase binding

Biological Process: DNA repair; gene expression; mRNA capping; nuclear mRNA splicing, via spliceosome; nucleotide-excision repair; positive regulation of RNA splicing; positive regulation of viral transcription; regulation of transcription, DNA-dependent; RNA elongation from RNA polymerase II promoter; RNA splicing; RNA-mediated gene silencing; somatic stem cell maintenance; transcription from RNA polymerase II promoter; transcription initiation from RNA polymerase II promoter; transcription termination; transcription-coupled nucleotide-excision repair; viral reproduction

NCBI Summary:This gene encodes the largest subunit of RNA polymerase II, the polymerase responsible for synthesizing messenger RNA in eukaryotes. The product of this gene contains a carboxy terminal domain composed of heptapeptide repeats that are essential for polymerase activity. These repeats contain serine and threonine residues that are phosphorylated in actively transcribing RNA polymerase. In addition, this subunit, in combination with several other polymerase subunits, forms the DNA binding domain of the polymerase, a groove in which the DNA template is transcribed into RNA. [provided by RefSeq, Jul 2008]
UniProt Code:P24928
NCBI GenInfo Identifier:281185484
NCBI Gene ID:5430
NCBI Accession:P24928.2
UniProt Secondary Accession:P24928,Q6NX41, A6NN93, B9EH88,
UniProt Related Accession:P24928
Molecular Weight:
NCBI Full Name:DNA-directed RNA polymerase II subunit RPB1
NCBI Synonym Full Names:polymerase (RNA) II subunit A
NCBI Official Symbol:POLR2A  
NCBI Official Synonym Symbols:RPB1; RPO2; POLR2; POLRA; RPBh1; RPOL2; RpIILS; hsRPB1; hRPB220  
NCBI Protein Information:DNA-directed RNA polymerase II subunit RPB1
UniProt Protein Name:DNA-directed RNA polymerase II subunit RPB1
UniProt Synonym Protein Names:DNA-directed RNA polymerase II subunit A; DNA-directed RNA polymerase III largest subunit; RNA-directed RNA polymerase II subunit RPB1 (EC:2.7.7.48)
UniProt Gene Name:POLR2A  
UniProt Entry Name:RPB1_HUMAN
ComponentQuantity
96-Well Cell Culture Clear-Bottom Microplate2 plates
10X TBS24 mL
Quenching Buffer24 mL
Blocking Buffer50 mL
15X Wash Buffer50 mL
Primary Antibody Diluent12 mL
100x Anti-Phospho Target Antibody 60 µL
100x Anti-Target Antibody60 µL
Anti-GAPDH Antibody60 µL
HRP-Conjugated Anti-Rabbit IgG Antibody12 mL
HRP-Conjugated Anti-Mouse IgG Antibody12 mL
SDS Solution12 mL
Stop Solution24 mL
Ready-to-Use Substrate12 mL
Crystal Violet Solution12 mL
Adhesive Plate Seals2 seals

The following materials and/or equipment are NOT provided in this kit but are necessary to successfully conduct the experiment:

  • Microplate reader able to measure absorbance at 450 nm and/or 595 nm for Crystal Violet Cell Staining (Optional)
  • Micropipettes with capability of measuring volumes ranging from 1 µL to 1 ml
  • 37% formaldehyde (Sigma Cat# F-8775) or formaldehyde from other sources
  • Squirt bottle, manifold dispenser, multichannel pipette reservoir or automated microplate washer
  • Graph paper or computer software capable of generating or displaying logarithmic functions
  • Absorbent papers or vacuum aspirator
  • Test tubes or microfuge tubes capable of storing ≥1 ml
  • Poly-L-Lysine (Sigma Cat# P4832 for suspension cells)
  • Orbital shaker (optional)
  • Deionized or sterile water

*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

StepProcedure
1.Seed 200 µL of 20,000 adherent cells in culture medium in each well of a 96-well plate. The plates included in the kit are sterile and treated for cell culture. For suspension cells and loosely attached cells, coat the plates with 100 µL of 10 µg/ml Poly-L-Lysine (not included) to each well of a 96-well plate for 30 minutes at 37 °C prior to adding cells.
2.Incubate the cells for overnight at 37 °C, 5% CO2.
3.Treat the cells as desired.
4.Remove the cell culture medium and rinse with 200 µL of 1x TBS, twice.
5.Fix the cells by incubating with 100 µL of Fixing Solution for 20 minutes at room temperature. The 4% formaldehyde is used for adherent cells and 8% formaldehyde is used for suspension cells and loosely attached cells.
6.Remove the Fixing Solution and wash the plate 3 times with 200 µL 1x Wash Buffer for five minutes each time with gentle shaking on the orbital shaker. The plate can be stored at 4 °C for a week.
7.Add 100 µL of Quenching Buffer and incubate for 20 minutes at room temperature.
8.Wash the plate 3 times with 1x Wash Buffer for 5 minutes each time.
9.Add 200 µL of Blocking Buffer and incubate for 1 hour at room temperature.
10. Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time.
11.Add 50 µL of 1x primary antibodies Anti-POLR2A (Phospho-Ser1619) Antibody, Anti-POLR2A Antibody and/or Anti-GAPDH Antibody) to the corresponding wells, cover with Parafilm and incubate for 16 hours (overnight) at 4 °C. If the target expression is known to be high, incubate for 2 hours at room temperature.
12.Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time.
13.Add 50 µL of 1x secondary antibodies (HRP-Conjugated AntiRabbit IgG Antibody or HRP-Conjugated Anti-Mouse IgG Antibody) to corresponding wells and incubate for 1.5 hours at room temperature.
14.Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time.
15.Add 50 µL of Ready-to-Use Substrate to each well and incubate for 30 minutes at room temperature in the dark.
16.Add 50 µL of Stop Solution to each well and read OD at 450 nm immediately using the microplate reader.

(Additional Crystal Violet staining may be performed if desired – details of this may be found in the kit technical manual.)

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