eEF2 (Phospho-Thr56) Colorimetric Cell-Based ELISA Kit

Product Name:	eEF2 (Phospho-Thr56) Colorimetric Cell-Based ELISA
Product Code:	CBCAB00224
ELISA Type:	Cell-Based
Target:	eEF2 (Phospho-Thr56)
Reactivity:	Human, Mouse, Rat
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nm
Format:	2 x 96-Well Microplates

The eEF2 (Phospho-Thr56) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect eEF2 protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated eEF2 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on eEF2 phosphorylation.

Qualitative determination of eEF2 (Phospho-Thr56) concentration is achieved by an indirect ELISA format. In essence, eEF2 (Phospho-Thr56) is captured by eEF2 (Phospho-Thr56)-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: 1938, UniProt ID: P13639, OMIM: 130610, Unigene: Hs.515070
Gene Symbol:	EEF2
Sub Type:	Phospho

UniProt Protein Function:	EEF2: a member of the GTP-binding translation elongation factor family. An essential factor for protein synthesis. Promotes the GTP-dependent translocation of the nascent protein chain from the A-site to the P-site of the ribosome. This protein is completely inactivated by eEF2 kinase phosphorylation. eEF2 kinase is normally dependent on Ca2+ ions and calmodulin. eEF2 kinase can also be activated by PKA in response to elevated cAMP levels, which are generally increased in stress- or starvation-related conditions. A variety of treatments known to raise intracellular Ca2+ or cAMP levels have been shown to result in increased phosphorylation of eEF2, and thus to inhibit peptide-chain elongation.
UniProt Protein Details:	Protein type:Translation elongation; Translation Chromosomal Location of Human Ortholog: 19p13.3 Cellular Component: polysome; membrane; cytoplasm; plasma membrane; cytosol; ribonucleoprotein complex; nucleus Molecular Function:GTPase activity; protein binding; GTP binding; protein kinase binding; translation elongation factor activity Biological Process: translational elongation; cellular protein metabolic process; translation; positive regulation of translation; peptidyl-diphthamide biosynthetic process from peptidyl-histidine; pathogenesis; hemopoietic progenitor cell differentiation; gene expression; post-translational protein modification Disease: Spinocerebellar Ataxia 26
NCBI Summary:	This gene encodes a member of the GTP-binding translation elongation factor family. This protein is an essential factor for protein synthesis. It promotes the GTP-dependent translocation of the nascent protein chain from the A-site to the P-site of the ribosome. This protein is completely inactivated by EF-2 kinase phosporylation. [provided by RefSeq, Jul 2008]
UniProt Code:	P13639
NCBI GenInfo Identifier:	119172
NCBI Gene ID:	1938
NCBI Accession:	P13639.4
UniProt Secondary Accession:	P13639,Q58J86, B2RMP5, D6W618,
UniProt Related Accession:	P13639
Molecular Weight:	858
NCBI Full Name:	Elongation factor 2
NCBI Synonym Full Names:	eukaryotic translation elongation factor 2
NCBI Official Symbol:	EEF2
NCBI Official Synonym Symbols:	EF2; EF-2; EEF-2; SCA26
NCBI Protein Information:	elongation factor 2; polypeptidyl-tRNA translocase
UniProt Protein Name:	Elongation factor 2
Protein Family:	Elongation factor
UniProt Gene Name:	EEF2
UniProt Entry Name:	EF2_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-eEF2 (Phospho-Thr56) Antibody, Anti-eEF2 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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신경면역학: CNS의 면역체계

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eEF2 (Phospho-Thr56) Colorimetric Cell-Based ELISA Kit

Description