Tyrosinase Colorimetric Cell-Based ELISA Kit (CBCAB00025)

Product Name:	Tyrosinase Colorimetric Cell-Based ELISA Kit
Product Code:	CBCAB00025
ELISA Type:	Cell-Based
Target:	Tyrosinase
Reactivity:	Human
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:	96-Well Microplate

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

Qualitative determination of Tyrosinase concentration is achieved by an indirect ELISA format. In essence, Tyrosinase is captured by Tyrosinase-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: 7299, UniProt ID: P14679, OMIM: 103470/203100/601800/606933/606952, Unigene: Hs.503555
Gene Symbol:	TYR
Sub Type:	None

UniProt Protein Function:	TYR: This is a copper-containing oxidase that functions in the formation of pigments such as melanins and other polyphenolic compounds. Catalyzes the rate-limiting conversions of tyrosine to DOPA, DOPA to DOPA-quinone and possibly 5,6-dihydroxyindole to indole-5,6 quinone. Increased expression after UVB irradiation. Belongs to the tyrosinase family. 2 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:	Protein type:Amino Acid Metabolism - tyrosine; EC 1.14.18.1; Membrane protein, integral; Cofactor and Vitamin Metabolism - riboflavin; Oxidoreductase Chromosomal Location of Human Ortholog: 11q14.3 Cellular Component: lysosome; perinuclear region of cytoplasm; cytoplasm; melanosome membrane; integral to membrane; melanosome; Golgi-associated vesicle Molecular Function:protein binding; copper ion binding; protein homodimerization activity; protein heterodimerization activity; monophenol monooxygenase activity Biological Process: cell proliferation; visual perception; thymus development; melanin biosynthetic process from tyrosine; eye pigment biosynthetic process Disease: Albinism, Ocular, With Sensorineural Deafness; Albinism, Oculocutaneous, Type Ib; Albinism, Oculocutaneous, Type Ia; Skin/hair/eye Pigmentation, Variation In, 3
NCBI Summary:	The enzyme encoded by this gene catalyzes the first 2 steps, and at least 1 subsequent step, in the conversion of tyrosine to melanin. The enzyme has both tyrosine hydroxylase and dopa oxidase catalytic activities, and requires copper for function. Mutations in this gene result in oculocutaneous albinism, and nonpathologic polymorphisms result in skin pigmentation variation. The human genome contains a pseudogene similar to the 3' half of this gene. [provided by RefSeq, Oct 2008]
UniProt Code:	P14679
NCBI GenInfo Identifier:	401235
NCBI Gene ID:	7299
NCBI Accession:	P14679.3
UniProt Secondary Accession:	P14679,Q15675, Q15676, Q15680, Q8TAK4, Q9BYY0, Q9BZX1
UniProt Related Accession:	P14679
Molecular Weight:	Calculated MW: 42kDa/60kDaObserved MW: 80kDa
NCBI Full Name:	Tyrosinase
NCBI Synonym Full Names:	tyrosinase
NCBI Official Symbol:	TYR
NCBI Official Synonym Symbols:	ATN; CMM8; OCA1; OCA1A; OCAIA; SHEP3
NCBI Protein Information:	tyrosinase; LB24-AB; SK29-AB; monophenol monooxygenase; oculocutaneous albinism IA; tumor rejection antigen AB
UniProt Protein Name:	Tyrosinase
UniProt Synonym Protein Names:	LB24-AB; Monophenol monooxygenase; SK29-AB; Tumor rejection antigen AB
Protein Family:	Tyrosinase
UniProt Gene Name:	TYR
UniProt Entry Name:	TYRO_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-Tyrosinase 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.)