eEF2K (Phospho-Ser366)Colorimetric Cell-Based ELISA Kit (CBCAB01241)

Product Name:	eEF2K (Phospho-Ser366)Colorimetric Cell-Based ELISA Kit
Product Code:	CBCAB01241
ELISA Type:	Cell-Based
Target:	eEF2K (Phospho-Ser366)
Reactivity:	Human, Rat
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nm
Format:	2 x 96-Well Microplates

The eEF2K (Phospho-Ser366) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect eEF2K protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated eEF2K in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on eEF2K phosphorylation.

Qualitative determination of eEF2K (Phospho-Ser366) concentration is achieved by an indirect ELISA format. In essence, eEF2K (Phospho-Ser366) is captured by eEF2K (Phospho-Ser366)-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: 29904, UniProt ID: O00418, OMIM: 606968, Unigene: Hs.633744
Gene Symbol:	EEF2K
Sub Type:	Phospho

UniProt Protein Function:	EEF2K: an atypical protein kinase of the Alpha family. Dependent on Ca2 ions and calmodulin. May play a key role linking cellular energy status and the inhibition of protein synthesis, a major consumer of metabolic energy. Phosphorylates and inactivates eEF2, resulting in the inhibition of peptide-chain elongation eEF2K is phosphorylated and activated by AMPK in response to elevated AMP levels, which increase during cellular stress and starvation. Regulated in response to a range of stimuli including TNF-alpha. Overexpressed in breast cancer cell lines and tumors. Activated in rat glioblastoma, and growth was inhibited by the drug Rottlerin. Activity induced in ischemic heart muscle
UniProt Protein Details:	Protein type:Protein kinase, Ser/Thr (non-receptor); Translation; Kinase, protein; Protein kinase, atypical; EC 2.7.11.20; ATYPICAL group; Alpha family; eEF2K subfamily Chromosomal Location of Human Ortholog: 16p12.2 Cellular Component: cytoplasm; cytosol Molecular Function:calmodulin binding; elongation factor-2 kinase activity; translation factor activity, nucleic acid binding; calcium ion binding; ATP binding; protein kinase activity Biological Process: translational elongation; regulation of protein amino acid autophosphorylation; protein amino acid autophosphorylation; insulin receptor signaling pathway
NCBI Summary:	This gene encodes a highly conserved protein kinase in the calmodulin-mediated signaling pathway that links activation of cell surface receptors to cell division. This kinase is involved in the regulation of protein synthesis. It phosphorylates eukaryotic elongation factor 2 (EEF2) and thus inhibits the EEF2 function. The activity of this kinase is increased in many cancers and may be a valid target for anti-cancer treatment. [provided by RefSeq, Jul 2008]
UniProt Code:	O00418
NCBI GenInfo Identifier:	254763289
NCBI Gene ID:	29904
NCBI Accession:	O00418.2
UniProt Secondary Accession:	O00418,Q8N588,
UniProt Related Accession:	O00418
Molecular Weight:	725
NCBI Full Name:	Eukaryotic elongation factor 2 kinase
NCBI Synonym Full Names:	eukaryotic elongation factor 2 kinase
NCBI Official Symbol:	EEF2K
NCBI Official Synonym Symbols:	eEF-2K; HSU93850
NCBI Protein Information:	eukaryotic elongation factor 2 kinase; eEF-2 kinase; elongation factor-2 kinase; eukaroytic elongation factor 2 kinase; eukaryotic elongation factor-2 kinase; calmodulin-dependent protein kinase III; calcium/calmodulin-dependent eukaryotic elongation factor 2 kinase; calcium/calmodulin-dependent eukaryotic elongation factor-2 kinase
UniProt Protein Name:	Eukaryotic elongation factor 2 kinase
UniProt Synonym Protein Names:	Calcium/calmodulin-dependent eukaryotic elongation factor 2 kinase
Protein Family:	Eukaryotic elongation factor 2 kinase
UniProt Gene Name:	EEF2K
UniProt Entry Name:	EF2K_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-eEF2K (Phospho-Ser366) Antibody, Anti-eEF2K 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.)