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MAP3K8 (Phospho-Thr290) Colorimetric Cell-Based ELISA Kit

SKU:
CBCAB01238
Product Type:
ELISA Kit
ELISA Type:
Cell Based Phospho Specific
Research Area:
Cell Cycle
Reactivity:
Human
Mouse
Rat
Detection Method:
Colorimetric
€599
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Description

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MAP3K8 (Phospho-Thr290)Colorimetric Cell-Based ELISA Kit

The MAP3K8 Phospho-Thr290 Colorimetric Cell-Based ELISA Kit from Assay Genie is a reliable and effective tool for studying the phosphorylation of MAP3K8 on threonine 290 in cell-based assays. This kit allows for the accurate detection of phosphorylated MAP3K8 in cell lysates, providing valuable insights into the cellular signaling pathways involved.MAP3K8, also known as Cot/Tpl-2, plays a critical role in immune responses, inflammation, and cancer development. Phosphorylation of MAP3K8 at threonine 290 is a key regulatory mechanism that affects its activity and downstream signaling events.

Understanding the phosphorylation status of MAP3K8 can help researchers unravel its role in various cellular processes and disease pathologies.With high sensitivity and specificity, the MAP3K8 Phospho-Thr290 Colorimetric Cell-Based ELISA Kit offers reliable and reproducible results, allowing researchers to confidently analyze phosphorylation events in cell-based experiments. Whether studying MAP3K8 signaling pathways or investigating potential therapeutic targets, this kit provides a valuable tool for advancing scientific research in this field.

Product Name:MAP3K8 (Phospho-Thr290) Colorimetric Cell-Based ELISA
Product Code:CBCAB01238
ELISA Type:Cell-Based
Target:MAP3K8 (Phospho-Thr290)
Reactivity:Human, Mouse, Rat
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nm
Format:2 x 96-Well Microplates

The MAP3K8 (Phospho-Thr290) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect MAP3K8 protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated MAP3K8 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on MAP3K8 phosphorylation.

Qualitative determination of MAP3K8 (Phospho-Thr290) concentration is achieved by an indirect ELISA format. In essence, MAP3K8 (Phospho-Thr290) is captured by MAP3K8 (Phospho-Thr290)-specific primary (1ø) antibodies while the HRP-conjugated secondary (2ø) antibodies bind the Fc region of the 1ø antibody. Through this binding, the HRP enzyme conjugated to the 2ø 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: 1326, UniProt ID: P41279, OMIM: 191195, Unigene: Hs.432453
Gene Symbol:MAP3K8
Sub Type:Phospho
UniProt Protein Function:Cot: an oncogenic Ser/Thr kinase of the STE group. Activates IkappaB kinases, thus inducing the nuclear translocation of NF-kappaB. Promotes the production of TNF-alpha and IL-2 during T lymphocyte activation. Interacts with NFkB-p105. Overexpressed and amplified in breast tumors. Viral insertions induce rat lymphomas and mouse mammary carcinomas. Isolated as a transforming factor in two cell lines. Mediates LPS activation of macrophages. 2 isoforms of the human protein are produced by alternative initiation.
UniProt Protein Details:

Protein type:Protein kinase, STE; Protein kinase, Ser/Thr (non-receptor); Kinase, protein; Oncoprotein; EC 2.7.11.25; STE group; STE-Unique family

Chromosomal Location of Human Ortholog: 10p11.23

Cellular Component: cytoplasm; cytosol

Molecular Function:protein serine/threonine kinase activity; protein binding; MAP kinase kinase kinase activity; magnesium ion binding; ATP binding; receptor signaling protein serine/threonine kinase activity

Biological Process: activation of MAPKK activity; apoptosis; regulation of cell differentiation; T cell costimulation; MAPKKK cascade; cell cycle; protein amino acid phosphorylation

Disease: Lung Cancer

NCBI Summary:This gene is an oncogene that encodes a member of the serine/threonine protein kinase family. The encoded protein localizes to the cytoplasm and can activate both the MAP kinase and JNK kinase pathways. This protein was shown to activate IkappaB kinases, and thus induce the nuclear production of NF-kappaB. This protein was also found to promote the production of TNF-alpha and IL-2 during T lymphocyte activation. This gene may also utilize a downstream in-frame translation start codon, and thus produce an isoform containing a shorter N-terminus. The shorter isoform has been shown to display weaker transforming activity. Alternate splicing results in multiple transcript variants that encode the same protein. [provided by RefSeq, Sep 2011]
UniProt Code:P41279
NCBI GenInfo Identifier:346644795
NCBI Gene ID:1326
NCBI Accession:NP_001231063.1
UniProt Secondary Accession:P41279,Q14275, Q5T855, Q9HC81, A8K2Q5, D3DRX1,
UniProt Related Accession:P41279
Molecular Weight:52,925 Da
NCBI Full Name:mitogen-activated protein kinase kinase kinase 8
NCBI Synonym Full Names:mitogen-activated protein kinase kinase kinase 8
NCBI Official Symbol:MAP3K8  
NCBI Official Synonym Symbols:COT; EST; ESTF; TPL2; MEKK8; Tpl-2; c-COT  
NCBI Protein Information:mitogen-activated protein kinase kinase kinase 8; proto-oncogene c-Cot; tumor progression locus 2; Ewing sarcoma transformant; cot (cancer Osaka thyroid) oncogene; proto-oncogene serine/threoine protein kinase
UniProt Protein Name:Mitogen-activated protein kinase kinase kinase 8
UniProt Synonym Protein Names:Cancer Osaka thyroid oncogene; Proto-oncogene c-Cot; Serine/threonine-protein kinase cot; Tumor progression locus 2
UniProt Gene Name:MAP3K8  
UniProt Entry Name:M3K8_HUMAN
ComponentQuantity
96-Well Cell Culture Clear-Bottom Microplate2 plates
10X TBS24 mL
Quenching Buffer24 mL
Blocking Buffer50 mL
15X Wash Buffer50 mL
Primary Antibody Diluent12 mL
100x Anti-Phospho Target Antibody 60 µL
100x Anti-Target Antibody60 µL
Anti-GAPDH Antibody60 µL
HRP-Conjugated Anti-Rabbit IgG Antibody12 mL
HRP-Conjugated Anti-Mouse IgG Antibody12 mL
SDS Solution12 mL
Stop Solution24 mL
Ready-to-Use Substrate12 mL
Crystal Violet Solution12 mL
Adhesive Plate Seals2 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.

StepProcedure
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-MAP3K8 (Phospho-Thr290) Antibody, Anti-MAP3K8 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.)

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