NFAT4 Colorimetric Cell-Based ELISA Kit (CBCAB00779)

Product Name:	NFAT4 Colorimetric Cell-Based ELISA Kit
Product Code:	CBCAB00779
ELISA Type:	Cell-Based
Target:	NFAT4
Reactivity:	Human, Mouse
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:	96-Well Microplate

The NFAT4 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect NFAT4 protein expression profile in cells. The kit can be used for measuring the relative amounts of NFAT4 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on NFAT4.

Qualitative determination of NFAT4 concentration is achieved by an indirect ELISA format. In essence, NFAT4 is captured by NFAT4-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: 4775, UniProt ID: Q12968, OMIM: 602698, Unigene: Hs.632209
Gene Symbol:	NFATC3
Sub Type:	None

UniProt Protein Function:	NFAT4: a transcription factor that plays a role in the inducible expression of cytokine genes in T cells. Plays a role in the regulation of gene expression in T cells and immature thymocytes. Six spliced isoforms have been identified. Isoform 1 is predominantly expressed in thymus, peripheral blood leukocytes and kidney. Isoform 2 is predominantly expressed in skeletal muscle and is also found in thymus, kidney, testis, spleen, prostate, ovary, small intestine, heart, placenta and pancreas. Isoform 3 is expressed in thymus and kidney. Isoform 4 is expressed in thymus and skeletal muscle.
UniProt Protein Details:	Protein type:DNA-binding; Transcription factor Chromosomal Location of Human Ortholog: 16q22.2 Cellular Component: nucleoplasm; cytoplasm; nucleolus; cytosol; nucleus Molecular Function:protein binding; chromatin binding Biological Process: transcription from RNA polymerase II promoter; regulation of transcription from RNA polymerase II promoter; patterning of blood vessels; heart development; cellular respiration; muscle cell development; innate immune response; positive regulation of transcription from RNA polymerase II promoter; smooth muscle cell differentiation; inflammatory response
NCBI Summary:	The product of this gene is a member of the nuclear factors of activated T cells DNA-binding transcription complex. This complex consists of at least two components: a preexisting cytosolic component that translocates to the nucleus upon T cell receptor (TCR) stimulation and an inducible nuclear component. Other members of this family participate to form this complex also. The product of this gene plays a role in the regulation of gene expression in T cells and immature thymocytes. Several transcript variants encoding distinct isoforms have been identified for this gene. [provided by RefSeq, Nov 2010]
UniProt Code:	Q12968
NCBI GenInfo Identifier:	9087155
NCBI Gene ID:	4775
NCBI Accession:	Q12968.1
UniProt Secondary Accession:	Q12968,O75211, Q14516, Q99840, Q99841, Q99842,
UniProt Related Accession:	Q12968
Molecular Weight:	1075
NCBI Full Name:	Nuclear factor of activated T-cells, cytoplasmic 3
NCBI Synonym Full Names:	nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 3
NCBI Official Symbol:	NFATC3
NCBI Official Synonym Symbols:	NFAT4; NFATX
NCBI Protein Information:	nuclear factor of activated T-cells, cytoplasmic 3; NF-ATc3; T cell transcription factor NFAT4; nuclear factor of activated T-cells c3 isoform IE-Xa
UniProt Protein Name:	Nuclear factor of activated T-cells, cytoplasmic 3
UniProt Synonym Protein Names:	NFATx; T-cell transcription factor NFAT4; NF-AT4
Protein Family:	Nuclear factor of activated T-cells
UniProt Gene Name:	NFATC3
UniProt Entry Name:	NFAC3_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-NFAT4 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.)