IRF2 Colorimetric Cell-Based ELISA

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SKU:
CBCAB00976
€499

Description

IRF2 Cell-Based ELISA Kit

The IRF2 Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can monitor IRF2 protein expression profile in cells. The kit can be used for measuring the relative amounts of IRF2 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on IRF2 .

How does our IRF2 Colorimetric Cell-Based ELISA Kit work?

Qualitative determination of IRF2 concentration is achieved by an indirect ELISA format. In essence, IRF2 is captured by IRF2 -specific primary (1°) antibodies while the HRP-conjugated secondary (2°) antibodies bind the Fc region of the 1° antibody. Through this binding, the HRP enzyme conjugated to the 2° 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.

IRF2 Colorimetric Cell-Based ELISA - Information

Product Name:IRF2 Colorimetric Cell-Based ELISA
Product Code:/SKUCBCAB00976
Description:The IRF2 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect IRF2 protein expression profile in cells. The kit can be used for measuring the relative amounts of IRF2 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on IRF2.
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nm
Storage/Stability:4°C/6 Months
Reactivity:Human, Mouse, Rat
Assay Type:Cell-Based
Database Links:Gene ID: 3660, UniProt ID: P14316, OMIM #: 147576, Unigene #: Hs.654566
Format:96-Well Microplate
NCBI Gene Symbol:IRF2
Sub Type:None
Target Name:IRF2

Kit Principle

Figure: Schematic representation of ELISA Genie Cell-Based Colorimetric ELISA principle

Kit components

Quantity

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 ul
100x Anti-Target Antibody60 ul
Anti-GAPDH Antibody60 ul
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

Additional equipment and materials required

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

Kit Protocol

This is a summarized version of the kit protocol. Please view the technical manual of this kit for information on sample preparation, reagent preparation and plate lay out.

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-IRF2 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.)

IRF2 - Protein Information

UniProt Protein Function:IRF2: Specifically binds to the upstream regulatory region of type I IFN and IFN-inducible MHC class I genes (the interferon consensus sequence (ICS)) and represses those genes. Also acts as an activator for several genes including H4 and IL7. Constitutively binds to the ISRE promoter to activate IL7. Involved in cell cycle regulation through binding the site II (HiNF-M) promoter region of H4 and activating transcription during cell growth. Antagonizes IRF1 transcriptional activation. Interacts with BRD7, IRF2BP1 and IRF2BP2. Interacts with CREBBP in growing cells; the interaction acetylates IRF2 and regulates IRF2-dependent H4 promoter activity. By viruses and IFN. Expressed throughout the epithelium of the colon. Also expressed in lamina propria. Belongs to the IRF family. 2 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:

Protein type:Transcription factor; DNA-binding

Chromosomal Location of Human Ortholog: 4q34.1-q35.1

Cellular Component: nucleoplasm; focal adhesion; cytoplasm; cytosol

Molecular Function:protein binding; DNA binding; transcription factor activity

Biological Process: cell proliferation; regulation of transcription, DNA-dependent; transcription, DNA-dependent; cytokine and chemokine mediated signaling pathway; positive regulation of transcription from RNA polymerase II promoter; negative regulation of transcription from RNA polymerase II promoter; blood coagulation

NCBI Summary:IRF2 encodes interferon regulatory factor 2, a member of the interferon regulatory transcription factor (IRF) family. IRF2 competitively inhibits the IRF1-mediated transcriptional activation of interferons alpha and beta, and presumably other genes that employ IRF1 for transcription activation. However, IRF2 also functions as a transcriptional activator of histone H4. [provided by RefSeq, Jul 2008]
UniProt Code:P14316
NCBI GenInfo Identifier:20141499
NCBI Gene ID:3660
NCBI Accession:P14316.2
UniProt Secondary Accession:P14316,Q6IAS7, Q96B99, D6RCK5, H0Y8S3,
UniProt Related Accession:P14316
Molecular Weight:39,156 Da
NCBI Full Name:Interferon regulatory factor 2
NCBI Synonym Full Names:interferon regulatory factor 2
NCBI Official Symbol:IRF2  
NCBI Official Synonym Symbols:IRF-2  
NCBI Protein Information:interferon regulatory factor 2
UniProt Protein Name:Interferon regulatory factor 2
Protein Family:Interferon regulatory factor
UniProt Gene Name:IRF2  
UniProt Entry Name:IRF2_HUMAN
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