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IRF-3 (Phospho-Ser396) Fluorometric Cell-Based ELISA Kit

SKU:
FBCAB00041
Product Type:
ELISA Kit
ELISA Type:
Cell Based Phospho Specific
Reactivity:
Human
Mouse
Rat
Detection Method:
Fluorometric
€599
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Description

Technical Manual

IRF-3 (Phospho-Ser396) Cell-Based ELISA Kit

The IRF-3 (Phospho-Ser396) Cell-Based ELISA Kit is a convenient, lysate- free, high throughput and sensitive assay kit that can monitor IRF-3 phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated IRF-3 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on IRF-3 phosphorylation.

How does our IRF-3 (Phospho-Ser396) Fluorometric Cell-Based ELISA Kit work?

Qualitative determination of IRF-3 (Phospho-Ser396) concentration is achieved by an indirect ELISA format. In essence, IRF-3 (Phospho-Ser396) is captured by IRF-3 (Phospho-Ser396)-specific primary (1°) antibodies while Dye 1-conjugated and Dye 2-conjugated secondary (2°) antibodies bind the Fc region of the 1° antibody. Through this binding, the dye conjugated to the 2° antibody can emit light at a certain wavelength given proper excitation, hence allowing for a fluorometric detection method. 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 RFU values.
2. An antibody against the nonphosphorylated counterpart of IRF-3 (Phospho-Ser396) is also provided for normalization purposes. The RFU values obtained for non-phosphorylated IRF-3 can be used to normalize the RFU value for phosphorylated IRF-3.

IRF-3 (Phospho-Ser396) Fluorometric Cell-Based ELISA Kit -Information

Product Name:IRF-3 (Phospho-Ser396) Fluorometric Cell-Based ELISA Kit
Product Code/SKU:FBCAB00041
Description:The IRF-3 (Phospho-Ser396) Fluorometric Cell-Based Phospho ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can monitor IRF-3 (Phospho-Ser396) protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated IRF-3 (Phospho-Ser396) in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals, or activators have on IRF3 phosphorylation.
Dynamic Range:> 5000 Cells
Detection Method:Fluorometric
Storage/Stability:4°C/6 Months
Reactivity:Human, Mouse, Rat
Assay Type:Cell-Based ELISA
Database Links:Gene ID: 3661, UniProt ID: Q14653, OMIM #: 603734, Unigene #: Hs.75254
Format:Two 96-Well Plates
NCBI Gene Symbol:IRF3
Sub Type:Phospho
Target Name:Phospho-IRF-3 (Ser396)

Kit Principle

Figure: Schematic representation of Assay Genie Cell-Based Fluorometric ELISA principle

Kit components

Quantity

96-Well Black Cell CultureClear-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 Antibody110 µl
Dye-1 Conjugated Anti-Rabbit IgG Antibody6 ml
Dye-2 Conjugated Anti-Mouse IgG Antibody6 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:

  • Fluorescent plate reader with two channels at Ex/Em: 651/667 and 495/521
  • Micropipettes capable of measuring volumes from 1 µl to 1 ml
  • Deionized or sterile water (ddH2O)
  • 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)

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 desired cell concentration in culture medium into each well of the 96-well plates. 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 3 minutes. 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 3 minutes each time.
9.Dispense 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 3 minutes each time.
11.Add 50 µl of Primary Antibody Mixture P to corresponding wells for IRF-3 (Phospho-Ser396) detection. Add 50 µl of Primary Antibody Mixture NP to the corresponding wells for total IRF-3 detection. Cover the plate 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 3 minutes each time.
13.Add 50 ul of Secondary Antibody Mixture to corresponding wells and incubate for 1.5 hours at room temperature in the dark.
14.Wash 3 times with 200 µl of 1x Wash Buffer for 3 minutes each time.
15.Read the plate(s) at Ex/Em: 651/667 (Dye 1) and 495/521 (Dye 2). Shield plates from direct light exposure.
16.Wash 3 times with 200 µl of 1x Wash Buffer for 5 minutes each time.

IRF-3 (Phospho-Ser396) - Protein Information

UniProt Protein Function:IRF3: interferon regulatory factor 3, a member of the interferon regulatory transcription factor (IRF) family. IRF3 is found in an inactive cytoplasmic form that upon serine/threonine phosphorylation forms a complex with CREBBP. This complex translocates to the nucleus and activates the transcription of interferons alpha and beta, as well as other interferon-induced genes.
UniProt Protein Details:

Protein type:DNA-binding; Transcription factor

Chromosomal Location of Human Ortholog: 19q13.3-q13.4

Cellular Component: nucleoplasm; cytoplasm; nucleus; cytosol

Molecular Function:identical protein binding; protein binding; protein homodimerization activity; DNA binding; transcription cofactor activity; transcription factor activity

Biological Process: transcription from RNA polymerase II promoter; negative regulation of interferon-beta biosynthetic process; positive regulation of I-kappaB kinase/NF-kappaB cascade; viral reproduction; apoptosis; MyD88-independent toll-like receptor signaling pathway; cytokine and chemokine mediated signaling pathway; toll-like receptor 3 signaling pathway; negative regulation of transcription from RNA polymerase II promoter; negative regulation of defense response to virus by host; response to exogenous dsRNA; positive regulation of interferon type I production; lipopolysaccharide-mediated signaling pathway; toll-like receptor signaling pathway; positive regulation of interferon-beta production; innate immune response; interferon type I biosynthetic process; defense response to virus; toll-like receptor 4 signaling pathway; response to DNA damage stimulus; negative regulation of interferon type I production; positive regulation of interferon-alpha production; positive regulation of cytokine secretion

Disease: Herpes Simplex Encephalitis, Susceptibility To, 7

NCBI Summary:This gene encodes a member of the interferon regulatory transcription factor (IRF) family. The encoded protein is found in an inactive cytoplasmic form that upon serine/threonine phosphorylation forms a complex with CREBBP. This complex translocates to the nucleus and activates the transcription of interferons alpha and beta, as well as other interferon-induced genes. Alternatively spliced transcript variants encoding multiple isoforms have been observed for this gene. [provided by RefSeq, Nov 2011]
UniProt Code:Q14653
NCBI GenInfo Identifier:2497442
NCBI Gene ID:3661
NCBI Accession:Q14653.1
UniProt Secondary Accession:Q14653,Q5FBY1, Q5FBY2, Q5FBY4, Q7Z5G6, A8K7L2, B2RAZ3
UniProt Related Accession:Q14653
Molecular Weight:12,330 Da
NCBI Full Name:Interferon regulatory factor 3
NCBI Synonym Full Names:interferon regulatory factor 3
NCBI Official Symbol:IRF3  
NCBI Protein Information:interferon regulatory factor 3
UniProt Protein Name:Interferon regulatory factor 3
Protein Family:Interferon regulatory factor
UniProt Gene Name:IRF3  
UniProt Entry Name:IRF3_HUMAN

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