MAP3K7 Colorimetric Cell-Based ELISA Kit (CBCAB00741)

Product Name:	MAP3K7 Colorimetric Cell-Based ELISA Kit
Product Code:	CBCAB00741
ELISA Type:	Cell-Based
Target:	MAP3K7
Reactivity:	Human, Mouse, Rat
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:	96-Well Microplate

The MAP3K7 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect MAP3K7 protein expression profile in cells. The kit can be used for measuring the relative amounts of MAP3K7 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on MAP3K7.

Qualitative determination of MAP3K7 concentration is achieved by an indirect ELISA format. In essence, MAP3K7 is captured by MAP3K7-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: 6885, UniProt ID: O43318, OMIM: 602614, Unigene: Hs.714773
Gene Symbol:	MAP3K7
Sub Type:	None

UniProt Protein Function:	TAK1: a protein kinase of the MLK family. Mediates signal transduction induced by TGF beta and morphogenetic protein (BMP), and controls a variety of cell functions including transcription regulation and apoptosis. In response to IL-1, forms a kinase complex including TRAF6, MAP3K7P1/TAB1 and MAP3K7P2/TAB2; this complex is required for the activation of nuclear factor kappa B. Also activates MAPK8/JNK, MAP2K4/MKK4, and thus plays a role in the cell response to environmental stresses. Four alternatively spliced isoforms have been reported. Three splice variant isoforms have been described.
UniProt Protein Details:	Protein type:Kinase, protein; Protein kinase, TKL; EC 2.7.11.25; Protein kinase, Ser/Thr (non-receptor); TKL group; MLK family; TAK1 subfamily Chromosomal Location of Human Ortholog: 6q15 Cellular Component: nucleoplasm; cytoplasm; plasma membrane; endosome membrane; IkappaB kinase complex; nucleus; cytosol Molecular Function:MAP kinase kinase activity; protein serine/threonine kinase activity; protein binding; MAP kinase kinase kinase activity; magnesium ion binding; ATP binding; protein kinase activity Biological Process: I-kappaB kinase/NF-kappaB cascade; positive regulation of JNK activity; activation of MAPKK activity; apoptosis; activation of MAPK activity; stress-activated MAPK cascade; positive regulation of T cell cytokine production; toll-like receptor 3 signaling pathway; T cell receptor signaling pathway; toll-like receptor 10 signaling pathway; activation of NF-kappaB transcription factor; toll-like receptor 5 signaling pathway; transforming growth factor beta receptor signaling pathway; JNK cascade; angiogenesis; toll-like receptor 4 signaling pathway; positive regulation of I-kappaB kinase/NF-kappaB cascade; transcription, DNA-dependent; MyD88-independent toll-like receptor signaling pathway; activation of NF-kappaB-inducing kinase; toll-like receptor 2 signaling pathway; MyD88-dependent toll-like receptor signaling pathway; positive regulation of interleukin-2 production; toll-like receptor signaling pathway; innate immune response; toll-like receptor 9 signaling pathway; positive regulation of T cell activation; I-kappaB phosphorylation; neural tube formation
NCBI Summary:	The protein encoded by this gene is a member of the serine/threonine protein kinase family. This kinase mediates the signaling transduction induced by TGF beta and morphogenetic protein (BMP), and controls a variety of cell functions including transcription regulation and apoptosis. In response to IL-1, this protein forms a kinase complex including TRAF6, MAP3K7P1/TAB1 and MAP3K7P2/TAB2; this complex is required for the activation of nuclear factor kappa B. This kinase can also activate MAPK8/JNK, MAP2K4/MKK4, and thus plays a role in the cell response to environmental stresses. Four alternatively spliced transcript variants encoding distinct isoforms have been reported. [provided by RefSeq, Jul 2008]
UniProt Code:	O43318
NCBI GenInfo Identifier:	12643557
NCBI Gene ID:	6885
NCBI Accession:	O43318.1
UniProt Secondary Accession:	O43318,O43317, O43319, Q5TDN2, Q5TDN3, Q5TDT7, Q9NTR3 Q9NZ70, B2RE27, E1P523,
UniProt Related Accession:	O43318
Molecular Weight:	67,196 Da
NCBI Full Name:	Mitogen-activated protein kinase kinase kinase 7
NCBI Synonym Full Names:	mitogen-activated protein kinase kinase kinase 7
NCBI Official Symbol:	MAP3K7
NCBI Official Synonym Symbols:	TAK1; MEKK7; TGF1a
NCBI Protein Information:	mitogen-activated protein kinase kinase kinase 7; TGF-beta activated kinase 1; TGF-beta-activated kinase 1; transforming growth factor-beta-activated kinase 1
UniProt Protein Name:	Mitogen-activated protein kinase kinase kinase 7
UniProt Synonym Protein Names:	Transforming growth factor-beta-activated kinase 1
Protein Family:	Mitogen-activated protein kinase kinase kinase
UniProt Gene Name:	MAP3K7
UniProt Entry Name:	M3K7_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-MAP3K7 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.)