FADD (Phospho-Ser191) Colorimetric Cell-Based ELISA Kit

Product Name:	FADD (Phospho-Ser191) Colorimetric Cell-Based ELISA
Product Code:	CBCAB01698
ELISA Type:	Cell-Based
Target:	FADD (Phospho-Ser191)
Reactivity:	Mouse
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nm
Format:	2 x 96-Well Microplates

The FADD (Phospho-Ser191) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect FADD protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated FADD in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on FADD phosphorylation.

Qualitative determination of FADD (Phospho-Ser191) concentration is achieved by an indirect ELISA format. In essence, FADD (Phospho-Ser191) is captured by FADD (Phospho-Ser191)-specific primary antibodies while the HRP-conjugated secondary antibodies bind the Fc region of the primary antibody. Through this binding, the HRP enzyme conjugated to the secondary 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: 14082, UniProt ID: Q61160, OMIM: None, Unigene: Mm.5126
Gene Symbol:	FADD
Sub Type:	Phospho

UniProt Protein Function:	FADD: an adaptor molecule that mediates apoptosis. Recruited through its C-terminal death domain by Fas-receptor, tumor necrosis factor receptor, TNFRSF25, and TRAIL-receptor, participating in the death signaling initiated by these receptors. Interaction with the receptors unmasks the N-terminal effector domain of this protein, which recruits caspase-8, and thereby activates the cysteine protease cascade. Knockout studies in mice also suggest the importance of this protein in early T cell development.
UniProt Protein Details:	Protein type:Adaptor/scaffold; Apoptosis Cellular Component: CD95 death-inducing signaling complex; cytoplasm; cytosol; lipid raft; neuron projection; plasma membrane; protein complex Molecular Function:death receptor binding; identical protein binding; protease binding; protein binding; protein complex binding; tumor necrosis factor receptor binding; tumor necrosis factor receptor superfamily binding Biological Process: apoptosis; cardiac muscle development; defense response to virus; innate immune response; lymph node development; positive regulation of activated T cell proliferation; positive regulation of adaptive immune response; positive regulation of apoptosis; positive regulation of interferon-gamma production; positive regulation of interleukin-8 production; positive regulation of macrophage differentiation; positive regulation of proteolysis; positive regulation of T cell mediated cytotoxicity; positive regulation of transcription from RNA polymerase II promoter; positive regulation of tumor necrosis factor production; protein heterooligomerization; spleen development; T cell differentiation; T cell differentiation in the thymus; T cell homeostasis; thymus development
UniProt Code:	Q61160
NCBI GenInfo Identifier:	2498356
NCBI Gene ID:	14082
NCBI Accession:	Q61160.1
UniProt Secondary Accession:	Q61160,Q3TC37, Q61082,
UniProt Related Accession:	Q61160
Molecular Weight:	22,960 Da
NCBI Full Name:	FAS-associated death domain protein
NCBI Synonym Full Names:	Fas (TNFRSF6)-associated via death domain
NCBI Official Symbol:	Fadd
NCBI Official Synonym Symbols:	Mort1/FADD
NCBI Protein Information:	FAS-associated death domain protein
UniProt Protein Name:	FAS-associated death domain protein
UniProt Synonym Protein Names:	FAS-associating death domain-containing protein; Mediator of receptor induced toxicity; Protein FADD
Protein Family:	Fas-associated death domain protein
UniProt Gene Name:	Fadd
UniProt Entry Name:	FADD_MOUSE

Component	Quantity
96-Well Cell Culture Clear-Bottom Microplate	2 plates
10X TBS	24 mL
Quenching Buffer	24 mL
Blocking Buffer	50 mL
15X Wash Buffer	50 mL
Primary Antibody Diluent	12 mL
100x Anti-Phospho Target Antibody	60 µL
100x Anti-Target Antibody	60 µL
Anti-GAPDH Antibody	60 µL
HRP-Conjugated Anti-Rabbit IgG Antibody	12 mL
HRP-Conjugated Anti-Mouse IgG Antibody	12 mL
SDS Solution	12 mL
Stop Solution	24 mL
Ready-to-Use Substrate	12 mL
Crystal Violet Solution	12 mL
Adhesive Plate Seals	2 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.

Step	Procedure
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-FADD (Phospho-Ser191) Antibody, Anti-FADD 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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신경면역학: CNS의 면역체계

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FADD (Phospho-Ser191) Colorimetric Cell-Based ELISA Kit

Description