FOXE3 Colorimetric Cell-Based ELISA

Product Name:	FOXE3 Colorimetric Cell-Based ELISA
Product Code:	CBCAB01086
ELISA Type:	Cell-Based
Target:	FOXE3
Reactivity:	Human, Mouse, Rat
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:	96-Well Microplate

The FOXE3 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect FOXE3 protein expression profile in cells. The kit can be used for measuring the relative amounts of FOXE3 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on FOXE3.

Qualitative determination of FOXE3 concentration is achieved by an indirect ELISA format. In essence, FOXE3 is captured by FOXE3-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: 2301, UniProt ID: Q13461, OMIM: 601094, Unigene: Hs.112968
Gene Symbol:	FOXE3
Sub Type:	None

UniProt Protein Function:	FOXE3: Defects in FOXE3 are a cause of anterior segment mesenchymal dysgenesis (ASMD); also known as anterior segment ocular dysgenesis (ASOD). ASMD consists of a range of developmental defects in structures at the front of the eye, resulting from abnormal migration or differentiation of the neural crest derived mesenchymal cells that give rise to the cornea, iris, and other components of the anterior chamber during eye development. Mature anterior segment anomalies are associated with an increased risk of glaucoma and corneal opacity. Conditions falling within the phenotypic spectrum include aniridia, posterior embryotoxon, Axenfeld anomaly, Reiger anomaly/syndrome, Peters anomaly, and iridogoniodysgenesis. Defects in FOXE3 are a cause of congenital primary aphakia (CPA). Aphakia is a rare congenital eye disorder in which the lens is missing. It has been histologically subdivided into primary and secondary forms, in accordance with the severity of defects of the ocular tissues, whose development requires the initial presence of a lens. CPA results from an early developmental arrest, around the 4th-5th week of gestation in humans, that prevents the formation of any lens structure and leads to severe secondary ocular defects, including a complete aplasia of the anterior segment of the eye. In contrast, in secondary aphakic eyes, lens induction has occurred, and the lens vesicle has developed to some degree but finally has progressively resorbed perinatally, leading, therefore, to less-severe ocular defects.
UniProt Protein Details:	Protein type:DNA-binding Chromosomal Location of Human Ortholog: 1p32 Cellular Component: nucleus; transcription factor complex Molecular Function:DNA binding; sequence-specific DNA binding; transcription factor activity Biological Process: anatomical structure morphogenesis; cell development; cell differentiation; lens development in camera-type eye; negative regulation of apoptosis; regulation of transcription from RNA polymerase II promoter; transcription from RNA polymerase II promoter Disease: Anterior Segment Mesenchymal Dysgenesis; Aphakia, Congenital Primary
NCBI Summary:	This intronless gene belongs to the forkhead family of transcription factors, which is characterized by a distinct forkhead domain. The protein encoded functions as a lens-specific transcription factor and plays an important role in vertebrate lens formation. Mutations in this gene are associated with anterior segment mesenchymal dysgenesis and congenital primary aphakia. [provided by RefSeq, Dec 2009]
UniProt Code:	Q13461
NCBI GenInfo Identifier:	12644406
NCBI Gene ID:	2301
NCBI Accession:	Q13461.2
UniProt Secondary Accession:	Q13461,Q5SVY9, Q9NQV9,
UniProt Related Accession:	Q13461
Molecular Weight:	33,234 Da
NCBI Full Name:	Forkhead box protein E3
NCBI Synonym Full Names:	forkhead box E3
NCBI Official Symbol:	FOXE3
NCBI Official Synonym Symbols:	FKHL12; FREAC8
NCBI Protein Information:	forkhead box protein E3
UniProt Protein Name:	Forkhead box protein E3
UniProt Synonym Protein Names:	Forkhead-related protein FKHL12; Forkhead-related transcription factor 8; FREAC-8
Protein Family:	Forkhead box protein
UniProt Gene Name:	FOXE3
UniProt Entry Name:	FOXE3_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-FOXE3 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.)

슈반 세포: 신경 기능을 지원하는 특수 세포

신경면역학: CNS의 면역체계

바르덴부르크 증후군 및 클라인-바르덴부르크 증후군

FOXE3 Colorimetric Cell-Based ELISA

Description