MYPT1 Colorimetric Cell-Based ELISA Kit (CBCAB00775)

Product Name:	MYPT1 Colorimetric Cell-Based ELISA Kit
Product Code:	CBCAB00775
ELISA Type:	Cell-Based
Target:	MYPT1
Reactivity:	Human, Mouse, Rat
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:	96-Well Microplate

The MYPT1 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect MYPT1 protein expression profile in cells. The kit can be used for measuring the relative amounts of MYPT1 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on MYPT1.

Qualitative determination of MYPT1 concentration is achieved by an indirect ELISA format. In essence, MYPT1 is captured by MYPT1-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: 4659, UniProt ID: O14974, OMIM: 602021, Unigene: Hs.49582
Gene Symbol:	PPP1R12A
Sub Type:	None

UniProt Protein Function:	MYPT1: myosin phosphatase target subunit 1, a regulatory subunit of protein phosphatase 1. Also called the myosin-binding subunit of myosin phosphatase. Myosin phosphatase regulates the interaction of actin and myosin downstream of the guanosine triphosphatase Rho. Four splice-variant isoforms have been described.
UniProt Protein Details:	Protein type:Motility/polarity/chemotaxis; Protein phosphatase, regulatory subunit Chromosomal Location of Human Ortholog: 12q15-q21 Cellular Component: A band; actin cytoskeleton; centrosome; contractile fiber; cytoplasm; cytosol; focal adhesion; kinetochore; nucleoplasm; Z disc Molecular Function:enzyme inhibitor activity; phosphatase regulator activity; phosphoprotein phosphatase activity; protein binding; protein kinase binding; signal transducer activity Biological Process: centrosome organization and biogenesis; G2/M transition of mitotic cell cycle; mitosis; mitotic cell cycle; negative regulation of catalytic activity; positive regulation of transcription from RNA polymerase II promoter; protein amino acid dephosphorylation; regulation of cell adhesion; regulation of nucleocytoplasmic transport; small GTPase mediated signal transduction
NCBI Summary:	Myosin phosphatase target subunit 1, which is also called the myosin-binding subunit of myosin phosphatase, is one of the subunits of myosin phosphatase. Myosin phosphatase regulates the interaction of actin and myosin downstream of the guanosine triphosphatase Rho. The small guanosine triphosphatase Rho is implicated in myosin light chain (MLC) phosphorylation, which results in contraction of smooth muscle and interaction of actin and myosin in nonmuscle cells. The guanosine triphosphate (GTP)-bound, active form of RhoA (GTP.RhoA) specifically interacted with the myosin-binding subunit (MBS) of myosin phosphatase, which regulates the extent of phosphorylation of MLC. Rho-associated kinase (Rho-kinase), which is activated by GTP. RhoA, phosphorylated MBS and consequently inactivated myosin phosphatase. Overexpression of RhoA or activated RhoA in NIH 3T3 cells increased phosphorylation of MBS and MLC. Thus, Rho appears to inhibit myosin phosphatase through the action of Rho-kinase. Several transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, Jan 2009]
UniProt Code:	O14974
NCBI GenInfo Identifier:	41017262
NCBI Gene ID:	4659
NCBI Accession:	O14974.1
UniProt Secondary Accession:	O14974,Q2NKL4, Q569H0, Q86WU3, Q8NFR6, Q9BYH0, B4DZ09 F8VWB4,
UniProt Related Accession:	O14974
Molecular Weight:	105,641 Da
NCBI Full Name:	Protein phosphatase 1 regulatory subunit 12A
NCBI Synonym Full Names:	protein phosphatase 1 regulatory subunit 12A
NCBI Official Symbol:	PPP1R12A
NCBI Official Synonym Symbols:	MBS; M130; MYPT1
NCBI Protein Information:	protein phosphatase 1 regulatory subunit 12A
UniProt Protein Name:	Protein phosphatase 1 regulatory subunit 12A
UniProt Synonym Protein Names:	Myosin phosphatase-targeting subunit 1; Myosin phosphatase target subunit 1; Protein phosphatase myosin-binding subunit
Protein Family:	Protein phosphatase 1 regulatory
UniProt Gene Name:	PPP1R12A
UniProt Entry Name:	MYPT1_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-MYPT1 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.)