MEK1 (Phospho-Thr291)Colorimetric Cell-Based ELISA Kit (CBCAB01567)

Product Name:	MEK1 (Phospho-Thr291)Colorimetric Cell-Based ELISA Kit
Product Code:	CBCAB01567
ELISA Type:	Cell-Based
Target:	MEK1 (Phospho-Thr291)
Reactivity:	Human, Mouse, Rat
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nm
Format:	2 x 96-Well Microplates

The MEK1 (Phospho-Thr291) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect MEK1 protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated MEK1 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on MEK1 phosphorylation.

Qualitative determination of MEK1 (Phospho-Thr291) concentration is achieved by an indirect ELISA format. In essence, MEK1 (Phospho-Thr291) is captured by MEK1 (Phospho-Thr291)-specific primary (1Ã¸) antibodies while the HRP-conjugated secondary (2Ã¸) antibodies bind the Fc region of the 1Ã¸ antibody. Through this binding, the HRP enzyme conjugated to the 2Ã¸ 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: 5604, UniProt ID: Q02750, OMIM: 115150/176872, Unigene: Hs.145442
Gene Symbol:	MAP2K1
Sub Type:	Phospho

UniProt Protein Function:	MEK1: a dual-specificity protein kinase of the STE7 kinase family. Phosphorylated and activated by Raf, Mos and Cot kinases. Phosphorylates a Thr and a Tyr residue in a Thr-Glu-Tyr sequence located in the activation loop of ERK1 and ERK2. An essential component of MAP kinase signal transduction pathways involved in many cellular processes such as proliferation, differentiation, transcription regulation and development.
UniProt Protein Details:	Protein type:EC 2.7.12.2; Protein kinase, dual-specificity (non-receptor); Kinase, protein; Protein kinase, STE; STE group; STE7 family Chromosomal Location of Human Ortholog: 15q22.1-q22.33 Cellular Component: dendrite cytoplasm; Golgi apparatus; microtubule; focal adhesion; mitochondrion; endoplasmic reticulum; early endosome; perikaryon; cell cortex; cytosol; axon; perinuclear region of cytoplasm; late endosome; cytoplasm; plasma membrane; microtubule organizing center; nucleus Molecular Function:MAP kinase kinase activity; protein serine/threonine kinase activity; protein serine/threonine kinase activator activity; protein binding; Ras GTPase binding; receptor signaling protein tyrosine phosphatase activity; protein-tyrosine kinase activity; protein serine/threonine/tyrosine kinase activity; mitogen-activated protein kinase kinase kinase binding; ATP binding; protein kinase activity Biological Process: axon guidance; peptidyl-tyrosine phosphorylation; activation of MAPKK activity; nerve growth factor receptor signaling pathway; activation of MAPK activity; protein heterooligomerization; negative regulation of homotypic cell-cell adhesion; response to glucocorticoid stimulus; stress-activated MAPK cascade; cell motility involved in cell locomotion; pathogenesis; toll-like receptor 3 signaling pathway; signal transduction; chemotaxis; toll-like receptor 10 signaling pathway; toll-like receptor 5 signaling pathway; neuron differentiation; negative regulation of cell proliferation; positive regulation of RNA elongation from RNA polymerase II promoter; small GTPase mediated signal transduction; vesicle transport along microtubule; response to axon injury; cell cycle arrest; toll-like receptor 4 signaling pathway; Golgi inheritance; epidermal growth factor receptor signaling pathway; mitosis; fibroblast growth factor receptor signaling pathway; MyD88-independent toll-like receptor signaling pathway; MAPKKK cascade; melanosome transport; toll-like receptor 2 signaling pathway; MyD88-dependent toll-like receptor signaling pathway; keratinocyte differentiation; cell proliferation; regulation of stress-activated MAPK cascade; regulation of vascular smooth muscle contraction; Ras protein signal transduction; insulin receptor signaling pathway; toll-like receptor signaling pathway; innate immune response; toll-like receptor 9 signaling pathway; response to oxidative stress; positive regulation of Ras protein signal transduction; cell motility; positive regulation of cell differentiation; vascular endothelial growth factor receptor signaling pathway; positive regulation of cell migration Disease: Noonan Syndrome 1; Cardiofaciocutaneous Syndrome 3
NCBI Summary:	The protein encoded by this gene is a member of the dual specificity protein kinase family, which acts as a mitogen-activated protein (MAP) kinase kinase. MAP kinases, also known as extracellular signal-regulated kinases (ERKs), act as an integration point for multiple biochemical signals. This protein kinase lies upstream of MAP kinases and stimulates the enzymatic activity of MAP kinases upon wide variety of extra- and intracellular signals. As an essential component of MAP kinase signal transduction pathway, this kinase is involved in many cellular processes such as proliferation, differentiation, transcription regulation and development. [provided by RefSeq, Jul 2008]
UniProt Code:	Q02750
NCBI GenInfo Identifier:	400274
NCBI Gene ID:	5604
NCBI Accession:	Q02750.2
UniProt Related Accession:	Q02750
Molecular Weight:	393
NCBI Full Name:	Dual specificity mitogen-activated protein kinase kinase 1
NCBI Synonym Full Names:	mitogen-activated protein kinase kinase 1
NCBI Official Symbol:	MAP2K1
NCBI Official Synonym Symbols:	CFC3; MEK1; MKK1; MAPKK1; PRKMK1
NCBI Protein Information:	dual specificity mitogen-activated protein kinase kinase 1; MEK 1; MAPKK 1; MAPK/ERK kinase 1; ERK activator kinase 1; protein kinase, mitogen-activated, kinase 1 (MAP kinase kinase 1)
UniProt Protein Name:	Dual specificity mitogen-activated protein kinase kinase 1
UniProt Synonym Protein Names:	ERK activator kinase 1; MAPK/ERK kinase 1; MEK 1
UniProt Gene Name:	MAP2K1
UniProt Entry Name:	MP2K1_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-MEK1 (Phospho-Thr291) Antibody, Anti-MEK1 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.)