TGF alpha Colorimetric Cell-Based ELISA Kit (CBCAB00368)

Product Name:	TGF alpha Colorimetric Cell-Based ELISA Kit
Product Code:	CBCAB00368
ELISA Type:	Cell-Based
Target:	TGF alpha
Reactivity:	Human, Mouse, Rat
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:	96-Well Microplate

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

Qualitative determination of TGF alpha concentration is achieved by an indirect ELISA format. In essence, TGF alpha is captured by TGF alpha-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: 7039, UniProt ID: P01135, OMIM: 190170, Unigene: Hs.170009
Gene Symbol:	TGFA
Sub Type:	None

UniProt Protein Function:	TGFalpha: TGF alpha is a mitogenic polypeptide that is able to bind to the EGF receptor/EGFR and to act synergistically with TGF beta to promote anchorage-independent cell proliferation in soft agar. Interacts with the PDZ domains of MAGI3, SDCBP and SNTA1. The interaction with SDCBP, is required for the targeting to the cell surface. In the endoplasmic reticulum, in its immature form (i.e. with a prosegment and lacking full N-glycosylation), interacts with CNIH. In the Golgi apparatus, may form a complex with CNIH and GORASP2. Interacts (via cytoplasmic C-terminal domain) with NKD2. Isoform 1, isoform 3 and isoform 4 are expressed in keratinocytes and tumor-derived cell lines. 5 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:	Protein type:Cell cycle regulation; Ligand, receptor tyrosine kinase; Membrane protein, integral; Motility/polarity/chemotaxis Chromosomal Location of Human Ortholog: 2p13 Cellular Component: extracellular space; cell surface; basolateral plasma membrane; perinuclear region of cytoplasm; integral to plasma membrane; plasma membrane; cytoplasmic vesicle; nucleus Molecular Function:protein binding; growth factor activity; glycoprotein binding; epidermal growth factor receptor binding Biological Process: response to drug; epidermal growth factor receptor signaling pathway; cell proliferation; positive regulation of epidermal growth factor receptor activity; wound healing; positive regulation of mitosis; activation of MAPK activity; angiogenesis; positive regulation of epithelial cell proliferation; negative regulation of apoptosis
NCBI Summary:	This gene encodes a growth factor that is a ligand for the epidermal growth factor receptor, which activates a signaling pathway for cell proliferation, differentiation and development. This protein may act as either a transmembrane-bound ligand or a soluble ligand. This gene has been associated with many types of cancers, and it may also be involved in some cases of cleft lip/palate. Alternatively spliced transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, Sep 2011]
UniProt Code:	P01135
NCBI GenInfo Identifier:	135689
NCBI Gene ID:	7039
NCBI Accession:	P01135.1
UniProt Secondary Accession:	P01135,Q15577, Q53SK7, Q9BS56, Q9UEI3, Q9UKM1, Q9UKM2 Q9UKM3, A8K286,
UniProt Related Accession:	P01135
Molecular Weight:	17,329 Da
NCBI Full Name:	Protransforming growth factor alpha
NCBI Synonym Full Names:	transforming growth factor, alpha
NCBI Official Symbol:	TGFA
NCBI Official Synonym Symbols:	TFGA
NCBI Protein Information:	protransforming growth factor alpha; TGF-alpha
UniProt Protein Name:	Protransforming growth factor alpha
UniProt Synonym Protein Names:	EGF-like TGF; ETGF; TGF type 1
Protein Family:	Transforming growth factor
UniProt Gene Name:	TGFA
UniProt Entry Name:	TGFA_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 alpha 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.)