4E-BP1 (Phospho-Thr69)Colorimetric Cell-Based ELISA Kit (CBCAB00405)

Product Name:	4E-BP1 (Phospho-Thr69)Colorimetric Cell-Based ELISA Kit
Product Code:	CBCAB00405
ELISA Type:	Cell-Based
Target:	4E-BP1 (Phospho-Thr69)
Reactivity:	Mouse, Rat
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nm
Format:	2 x 96-Well Microplates

The 4E-BP1 (Phospho-Thr69) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect 4E-BP1 protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated 4E-BP1 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on 4E-BP1 phosphorylation.

Qualitative determination of 4E-BP1 (Phospho-Thr69) concentration is achieved by an indirect ELISA format. In essence, 4E-BP1 (Phospho-Thr69) is captured by 4E-BP1 (Phospho-Thr69)-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: 13685, UniProt ID: Q60876, OMIM: None, Unigene: Mm.6700
Gene Symbol:	EIF4EBP1
Sub Type:	Phospho

UniProt Protein Function:	4E-BP1: binds to eIF4E, preventing its assembly into the EIF4F complex and inhibiting cap-dependent translation. Phosphorylation of 4E-BP1 disrupts this binding, activating cap-dependent translation. Mediates the regulation of protein translation by hormones, growth factors and other stimuli that signal through the PI3 kinase pathway.
UniProt Protein Details:	Protein type:Translation; Translation initiation Chromosomal Location of Human Ortholog: 8p12 Cellular Component: nucleoplasm; protein complex; cytoplasm; cytosol Molecular Function:protein binding; translation repressor activity; eukaryotic initiation factor 4E binding Biological Process: negative regulation of translational initiation; response to ethanol; cellular protein metabolic process; TOR signaling pathway; translation; insulin receptor signaling pathway; translational initiation; gene expression; positive regulation of mitotic cell cycle; negative regulation of protein complex assembly; G1/S transition of mitotic cell cycle; lung development
NCBI Summary:	This gene encodes one member of a family of translation repressor proteins. The protein directly interacts with eukaryotic translation initiation factor 4E (eIF4E), which is a limiting component of the multisubunit complex that recruits 40S ribosomal subunits to the 5' end of mRNAs. Interaction of this protein with eIF4E inhibits complex assembly and represses translation. This protein is phosphorylated in response to various signals including UV irradiation and insulin signaling, resulting in its dissociation from eIF4E and activation of mRNA translation. [provided by RefSeq, Jul 2008]
UniProt Code:	Q60876
NCBI GenInfo Identifier:	4758258
NCBI Gene ID:	1978
NCBI Accession:	NP_004086.1
UniProt Secondary Accession:	Q60876,Q60876, Q62622,
UniProt Related Accession:	Q13541
Molecular Weight:
NCBI Full Name:	eukaryotic translation initiation factor 4E-binding protein 1
NCBI Synonym Full Names:	eukaryotic translation initiation factor 4E binding protein 1
NCBI Official Symbol:	EIF4EBP1
NCBI Official Synonym Symbols:	BP-1; 4EBP1; 4E-BP1; PHAS-I
NCBI Protein Information:	eukaryotic translation initiation factor 4E-binding protein 1
UniProt Protein Name:	Eukaryotic translation initiation factor 4E-binding protein 1
UniProt Synonym Protein Names:	Phosphorylated heat- and acid-stable protein regulated by insulin 1
UniProt Gene Name:	EIF4EBP1
UniProt Entry Name:	4EBP1_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-4E-BP1 (Phospho-Thr69) Antibody, Anti-4E-BP1 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.)