SEK1/MKK4 (Phospho-Thr261)Colorimetric Cell-Based ELISA Kit (CBCAB00107)

Product Name:	SEK1/MKK4 (Phospho-Thr261)Colorimetric Cell-Based ELISA Kit
Product Code:	CBCAB00107
ELISA Type:	Cell-Based
Target:	SEK1/MKK4 (Phospho-Thr261)
Reactivity:	Human, Mouse, Rat
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nm
Format:	2 x 96-Well Microplates

The SEK1/MKK4 (Phospho-Thr261) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect SEK1/MKK4 protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated SEK1/MKK4 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on SEK1/MKK4 phosphorylation.

Qualitative determination of SEK1/MKK4 (Phospho-Thr261) concentration is achieved by an indirect ELISA format. In essence, SEK1/MKK4 (Phospho-Thr261) is captured by SEK1/MKK4 (Phospho-Thr261)-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: 6416, UniProt ID: P45985, OMIM: 601335, Unigene: Hs.514681
Gene Symbol:	MAP2K4
Sub Type:	Phospho

UniProt Protein Function:	MKK4: dual specificity kinase of the STE7 family that phosphorylates and activates JNK1 and -2 as well as p38 but not ERK1 or -2. Mediates cellular responses to various cellular stresses and inflammatory cytokines. Phosphorylation by Akt inhibits MKK4 and suppresses stress-activated signal transduction.
UniProt Protein Details:	Protein type:Protein kinase, STE; Protein kinase, dual-specificity (non-receptor); Kinase, protein; EC 2.7.12.2; STE group; STE7 family Chromosomal Location of Human Ortholog: 17p12 Cellular Component: dendrite cytoplasm; axon; perikaryon; intracellular; cytosol; nucleus Molecular Function:protein serine/threonine kinase activity; protein binding; protein-tyrosine kinase activity; mitogen-activated protein kinase kinase kinase binding; JUN kinase kinase activity; ATP binding; protein kinase activity Biological Process: peptidyl-tyrosine phosphorylation; apoptosis; MyD88-independent toll-like receptor signaling pathway; stress-activated MAPK cascade; pathogenesis; toll-like receptor 3 signaling pathway; signal transduction; activation of JNK activity; toll-like receptor 10 signaling pathway; toll-like receptor 2 signaling pathway; toll-like receptor 5 signaling pathway; MyD88-dependent toll-like receptor signaling pathway; positive regulation of neuron apoptosis; toll-like receptor signaling pathway; JNK cascade; innate immune response; toll-like receptor 9 signaling pathway; toll-like receptor 4 signaling pathway; positive regulation of DNA replication; positive regulation of nitric-oxide synthase biosynthetic process
NCBI Summary:	This gene encodes a member of the mitogen-activated protein kinase (MAPK) family. Members of this family act as an integration point for multiple biochemical signals and are involved in a wide variety of cellular processes such as proliferation, differentiation, transcription regulation, and development. They form a three-tiered signaling module composed of MAPKKKs, MAPKKs, and MAPKs. This protein is phosphorylated at serine and threonine residues by MAPKKKs and subsequently phosphorylates downstream MAPK targets at threonine and tyrosine residues. A similar protein in mouse has been reported to play a role in liver organogenesis. A pseudogene of this gene is located on the long arm of chromosome X. Alternative splicing results in multiple transcript variants. [provided by RefSeq, Jul 2013]
UniProt Code:	P45985
NCBI GenInfo Identifier:	1170596
NCBI Gene ID:	6416
NCBI Accession:	P45985.1
UniProt Secondary Accession:	P45985,Q5U0B8, Q6FHX4, Q6P9H2, Q6PIE6, B2R7N7, B3KYB2 D3DTS5,
UniProt Related Accession:	P45985
Molecular Weight:	399
NCBI Full Name:	Dual specificity mitogen-activated protein kinase kinase 4
NCBI Synonym Full Names:	mitogen-activated protein kinase kinase 4
NCBI Official Symbol:	MAP2K4
NCBI Official Synonym Symbols:	JNKK; MEK4; MKK4; SEK1; SKK1; JNKK1; SERK1; MAPKK4; PRKMK4; SAPKK1; SAPKK-1
NCBI Protein Information:	dual specificity mitogen-activated protein kinase kinase 4; MEK 4; MAPKK 4; MAPK/ERK kinase 4; SAPK/ERK kinase 1; MAP kinase kinase 4; JNK-activated kinase 1; JNK-activating kinase 1; c-Jun N-terminal kinase kinase 1; stress-activated protein kinase kinase 1
UniProt Protein Name:	Dual specificity mitogen-activated protein kinase kinase 4
UniProt Synonym Protein Names:	JNK-activating kinase 1; MAPK/ERK kinase 4; MEK 4; SAPK/ERK kinase 1; SEK1; Stress-activated protein kinase kinase 1; SAPK kinase 1; SAPKK-1; SAPKK1; c-Jun N-terminal kinase kinase 1; JNKK
Protein Family:	Dual specificity mitogen-activated protein kinase kinase
UniProt Gene Name:	MAP2K4
UniProt Entry Name:	MP2K4_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-SEK1/MKK4 (Phospho-Thr261) Antibody, Anti-SEK1/MKK4 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.)