Factor Vlll Colorimetric Cell-Based ELISA

Introduction

The Colorimetric Cell-Based ELISA Kit allows for the detection of various target proteins and the effects that certain stimulation conditions have on target protein expression in different cell lines. Qualitative determination of target protein concentration is achieved by an indirect ELISA format. In essence, the target protein is captured by target-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 described: 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 is used to determine cell density. After staining, the results can be analyzed by normalizing the absorbance values to cell amounts, by which the plating difference can be adjusted. 3) If a phosphorylated target is being detected, an antibody against the nonphosphorylated counterpart will be provided for normalization purposes. The absorbance values obtained for the non-phosphorylated target can be used to normalize the absorbance values for the phosphorylated target. used to normalize the absorbance values for the phosphorylated target.

 

Overview

Product Name	Factor Vlll Colorimetric Cell-Based ELISA
SKU/Catalog Number	EKC1933
Storage & Handling	Store at 4°C for 6 months, at -20°C for 12 months. Avoid multiple freeze-thaw cycles(Shipped with wet ice.)
Size	100ul
Description	The Factor Vlll Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can monitor Factor Vlll protein expression profile in cells. The kit can be used for measuring the relative amounts of Factor Vlll in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on Factor Vlll.
Cite This Product	Factor Vlll Colorimetric Cell-Based ELISA (Boster Biological Technology, Pleasanton CA, USA, Catalog # EKC1933)
Sample Type	Cells.
Sensitivity	>5000 cells
Assay Range	> 5000 Cells
Reactivity	Human, Mouse

Assay Details

Kit Components

Reagent	Quantity	Container
96-Well Cell Culture Clear-Bottom Microplate	1 Plate	-
10x TBS	24 ml (10x)	Clear
Quenching Buffer	24 ml (1x)	Clear
Blocking Buffer	50 ml (1x)	Clear
15x Wash Buffer	50 ml (15x)	Clear
100x Anti-Factor Vlll Antibody (Rabbit Polyclonal)	60 µl (100x)	Purple
100x Anti-GAPDH Antibody (Mouse Monoclonal)	60 µl (100x)	Green
HRP-Conjugated Anti-Rabbit IgG Antibody	6 ml (1x)	Glass
HRP-Conjugated Anti-Mouse IgG Antibody	6 ml (1x)	Glass
Primary Antibody Diluent	12 ml (1x)	Clear
Ready-to-Use Substrate	12 ml (1x)	Brown
Stop Solution	12 ml (1x)	Clear
Crystal Violet Solution	6 ml (1x)	Glass
SDS Solution	24 ml (1x)	Clear
Adhesive Plate Seals	2 Seals	-

Materials Required But Not Provided

• 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 ul to 1 ml
• 37% formaldehyde (Sigma Cat# F-8775) or formaldehyde from other sources
• Deionized or sterile water
• 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
• Orbital shaker
• Poly-L-Lysine (Sigma Cat# P4832 for suspension cells)

Uniprot Id	P00451
Gene Name	F8
Protein Name	Coagulation factor VIII
Alternative Names	Coagulation factor VIII;Antihemophilic factor;AHF;Procoagulant component;Factor VIIIa heavy chain, 200 kDa isoform;Factor VIIIa heavy chain, 92 kDa isoform;Factor VIII B chain;Factor VIIIa light chain;F8;F8C;
Subcellular Localization	Secreted, extracellular space.
Molecular Weight	267009 MW

*if product is indicated to react with multiple species, protein info is based on the human gene.

Ontology

Protein Function	Factor VIII, along with calcium and phospholipid, acts as a cofactor for F9/factor IXa when it converts F10/factor X to the activated form, factor Xa.