MYT1 Colorimetric Cell-Based ELISA Kit (CBCAB00776)

Product Name:	MYT1 Colorimetric Cell-Based ELISA Kit
Product Code:	CBCAB00776
ELISA Type:	Cell-Based
Target:	MYT1
Reactivity:	Human
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:	96-Well Microplate

The MYT1 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect MYT1 protein expression profile in cells. The kit can be used for measuring the relative amounts of MYT1 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on MYT1.

Qualitative determination of MYT1 concentration is achieved by an indirect ELISA format. In essence, MYT1 is captured by MYT1-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: 9088, UniProt ID: Q99640, OMIM: 602474, Unigene: Hs.77783
Gene Symbol:	PKMYT1
Sub Type:	None

UniProt Protein Function:	Myt1: a dual specificity protein kinase of the WEE family. Preferentially phosphorylates and inactivates cell division cycle 2 protein (CDC2), and thus negatively regulates cell cycle G2/M transition. This kinase is associated with the membrane throughout the cell cycle. Its activity is highly regulated during the cell cycle. Alternative splice-variant isoforms have been reported.
UniProt Protein Details:	Protein type:Kinase, protein; Protein kinase, Other; Protein kinase, dual-specificity (non-receptor); EC 2.7.11.1; Other group; WEE family Chromosomal Location of Human Ortholog: 16p13.3 Cellular Component: cytosol; endoplasmic reticulum; endoplasmic reticulum membrane; Golgi apparatus; Golgi membrane; membrane; nucleoplasm Molecular Function:ATP binding; kinase activity; metal ion binding; protein binding; protein kinase activity; protein serine/threonine kinase activity Biological Process: G1/S transition of mitotic cell cycle; G2/M transition of mitotic cell cycle; mitosis; mitotic cell cycle; protein amino acid phosphorylation; regulation of cell cycle; regulation of cyclin-dependent protein kinase activity; regulation of mitosis
NCBI Summary:	This gene encodes a member of the serine/threonine protein kinase family. The encoded protein is a membrane-associated kinase that negatively regulates the G2/M transition of the cell cycle by phosphorylating and inactivating cyclin-dependent kinase 1. The activity of the encoded protein is regulated by polo-like kinase 1. Alternatively spliced transcript variants encoding multiple isoforms have been observed for this gene. [provided by RefSeq, May 2012]
UniProt Code:	Q99640
NCBI GenInfo Identifier:	55976573
NCBI Gene ID:	9088
NCBI Accession:	Q99640.1
UniProt Secondary Accession:	Q99640,O14731, Q7LE24, Q8TCM9, B3KUN8, B4DXD4, D3DUA4 F8W164, I3L1V2,
UniProt Related Accession:	Q99640
Molecular Weight:	47,284 Da
NCBI Full Name:	Membrane-associated tyrosine- and threonine-specific cdc2-inhibitory kinase
NCBI Synonym Full Names:	protein kinase, membrane associated tyrosine/threonine 1
NCBI Official Symbol:	PKMYT1
NCBI Official Synonym Symbols:	MYT1; PPP1R126
NCBI Protein Information:	membrane-associated tyrosine- and threonine-specific cdc2-inhibitory kinase
UniProt Protein Name:	Membrane-associated tyrosine- and threonine-specific cdc2-inhibitory kinase
UniProt Synonym Protein Names:	Myt1 kinase
Protein Family:	Membrane-associated tyrosine- and threonine-specific cdc2-inhibitory kinase
UniProt Gene Name:	PKMYT1
UniProt Entry Name:	PMYT1_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-MYT1 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.)