Coagulation Factor III (Phospho-Ser290) Colorimetric Cell-Based ELISA Kit

Product Name:	Coagulation Factor III (Phospho-Ser290) Colorimetric Cell-Based ELISA
Product Code:	CBCAB01406
ELISA Type:	Cell-Based
Target:	Coagulation Factor III (Phospho-Ser290)
Reactivity:	Human
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nm
Format:	2 x 96-Well Microplates

The Coagulation Factor III (Phospho-Ser290) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect Coagulation Factor III protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated Coagulation Factor III in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on Coagulation Factor III phosphorylation.

Qualitative determination of Coagulation Factor III (Phospho-Ser290) concentration is achieved by an indirect ELISA format. In essence, Coagulation Factor III (Phospho-Ser290) is captured by Coagulation Factor III (Phospho-Ser290)-specific primary (1Ãƒ¸) antibodies while the HRP-conjugated secondary (2Ãƒ¸) antibodies bind the Fc region of the 1Ãƒ¸ antibody. Through this binding, the HRP enzyme conjugated to the 2Ãƒ¸ 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: 2152, UniProt ID: P13726, OMIM: 134390, Unigene: Hs.62192
Gene Symbol:	F3
Sub Type:	Phospho

UniProt Protein Function:	F3: Initiates blood coagulation by forming a complex with circulating factor VII or VIIa. The [TF:VIIa] complex activates factors IX or X by specific limited protolysis. TF plays a role in normal hemostasis by initiating the cell-surface assembly and propagation of the coagulation protease cascade. Interacts with HSPE; the interaction, inhibited by heparin, promotes the generation of activated factor X and activates coagulation in the presence of activated factor VII. TF expression is highly dependent upon cell type. TF can also be induced by the inflammatory mediators interleukin 1 and TNF-alpha, as well as by endotoxin, to appear on monocytes and vascular endothelial cells as a component of cellular immune response. Lung, placenta and pancreas. Belongs to the tissue factor family. 2 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:	Protein type:Membrane protein, integral; Cell surface Chromosomal Location of Human Ortholog: 1p22-p21 Cellular Component: cell surface; cytoplasm; extracellular matrix; extracellular space; integral to membrane; plasma membrane Molecular Function:hematopoietin/interferon-class (D200-domain) cytokine receptor activity; phospholipid binding; protease binding; protein binding Biological Process: activation of blood coagulation via clotting cascade; activation of plasma proteins during acute inflammatory response; aging; blood coagulation; blood coagulation, extrinsic pathway; caspase activation; cytokine and chemokine mediated signaling pathway; positive regulation of angiogenesis; positive regulation of cell migration; positive regulation of endothelial cell proliferation; positive regulation of positive chemotaxis; positive regulation of protein kinase B signaling cascade; positive regulation of smooth muscle cell migration; response to estradiol stimulus; response to lipopolysaccharide; response to low density lipoprotein stimulus; response to mechanical stimulus; response to temperature stimulus
NCBI Summary:	This gene encodes coagulation factor III which is a cell surface glycoprotein. This factor enables cells to initiate the blood coagulation cascades, and it functions as the high-affinity receptor for the coagulation factor VII. The resulting complex provides a catalytic event that is responsible for initiation of the coagulation protease cascades by specific limited proteolysis. Unlike the other cofactors of these protease cascades, which circulate as nonfunctional precursors, this factor is a potent initiator that is fully functional when expressed on cell surfaces. There are 3 distinct domains of this factor: extracellular, transmembrane, and cytoplasmic. This protein is the only one in the coagulation pathway for which a congenital deficiency has not been described. Alternate splicing results in multiple transcript variants.[provided by RefSeq, May 2010]
UniProt Code:	P13726
NCBI GenInfo Identifier:	135666
NCBI Gene ID:	2152
NCBI Accession:	P13726.1
UniProt Secondary Accession:	P13726,Q6FHG2, Q86WH4, D3DT47,
UniProt Related Accession:	P13726
Molecular Weight:	27,145 Da
NCBI Full Name:	Tissue factor
NCBI Synonym Full Names:	coagulation factor III, tissue factor
NCBI Official Symbol:	F3
NCBI Official Synonym Symbols:	TF; TFA; CD142
NCBI Protein Information:	tissue factor
UniProt Protein Name:	Tissue factor
UniProt Synonym Protein Names:	Coagulation factor III; Thromboplastin; CD_antigen: CD142
Protein Family:	Tissue factor
UniProt Gene Name:	F3
UniProt Entry Name:	TF_HUMAN

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-Coagulation Factor III (Phospho-Ser290) Antibody, Anti-Coagulation Factor III 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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Coagulation Factor III (Phospho-Ser290) Colorimetric Cell-Based ELISA Kit

Description