TGF beta3 Colorimetric Cell-Based ELISA Kit (CBCAB00883)

Product Name:	TGF beta3 Colorimetric Cell-Based ELISA Kit
Product Code:	CBCAB00883
ELISA Type:	Cell-Based
Target:	TGF beta3
Reactivity:	Human, Mouse, Rat
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:	96-Well Microplate

The TGF beta3 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect TGF beta3 protein expression profile in cells. The kit can be used for measuring the relative amounts of TGF beta3 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on TGF beta3.

Qualitative determination of TGF beta3 concentration is achieved by an indirect ELISA format. In essence, TGF beta3 is captured by TGF beta3-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: 7043, UniProt ID: P10600, OMIM: 107970/190230, Unigene: Hs.592317
Gene Symbol:	TGFB3
Sub Type:	None

UniProt Protein Function:	TGFB3: Involved in embryogenesis and cell differentiation. Homodimer; disulfide-linked. Interacts with ASPN. Belongs to the TGF-beta family.
UniProt Protein Details:	Protein type:Ligand, receptor tyrosine kinase; Motility/polarity/chemotaxis; Secreted; Cell development/differentiation; Secreted, signal peptide Chromosomal Location of Human Ortholog: 14q24 Cellular Component: extracellular matrix; extracellular region; extracellular space; plasma membrane Molecular Function:cytokine activity; identical protein binding; protein binding; punt binding; transforming growth factor beta binding Biological Process: alveolus development; in utero embryonic development; intercellular junction assembly and maintenance; mammary gland development; negative regulation of cell proliferation; negative regulation of DNA replication; negative regulation of neuron apoptosis; palate development; platelet degranulation; positive regulation of apoptosis; positive regulation of bone mineralization; positive regulation of collagen biosynthetic process; positive regulation of DNA replication; positive regulation of filopodium formation; positive regulation of protein secretion; positive regulation of transcription from RNA polymerase II promoter; positive regulation of transcription, DNA-dependent; regulation of apoptosis; regulation of cell proliferation; regulation of MAPKKK cascade; response to hypoxia; response to progesterone stimulus; salivary gland morphogenesis; transforming growth factor beta receptor signaling pathway; uterine wall breakdown Disease: Arrhythmogenic Right Ventricular Dysplasia, Familial, 1; Rienhoff Syndrome
NCBI Summary:	This gene encodes a member of the TGF-beta family of proteins. The encoded protein is secreted and is involved in embryogenesis and cell differentiation. Defects in this gene are a cause of familial arrhythmogenic right ventricular dysplasia 1. [provided by RefSeq, Mar 2009]
UniProt Code:	P10600
NCBI GenInfo Identifier:	135684
NCBI Gene ID:	7043
NCBI Accession:	P10600.1
UniProt Secondary Accession:	P10600,Q8WV88,
UniProt Related Accession:	P10600
Molecular Weight:	35,708 Da
NCBI Full Name:	Transforming growth factor beta-3
NCBI Synonym Full Names:	transforming growth factor beta 3
NCBI Official Symbol:	TGFB3
NCBI Official Synonym Symbols:	ARVD; LDS5; RNHF; ARVD1; TGF-beta3
NCBI Protein Information:	transforming growth factor beta-3
UniProt Protein Name:	Transforming growth factor beta-3
Protein Family:	Transforming growth factor
UniProt Gene Name:	TGFB3
UniProt Entry Name:	TGFB3_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-TGF beta3 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.)