PEA-15 (Phospho-Ser116) Colorimetric Cell-Based ELISA Kit

Product Name:	PEA-15 (Phospho-Ser116)Colorimetric Cell-Based ELISA Kit
Product Code:	CBCAB01311
ELISA Type:	Cell-Based
Target:	PEA-15 (Phospho-Ser116)
Reactivity:	Human, Mouse, Rat
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nm
Format:	2 x 96-Well Microplates

The PEA-15 (Phospho-Ser116) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect PEA-15 protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated PEA-15 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on PEA-15 phosphorylation.

Qualitative determination of PEA-15 (Phospho-Ser116) concentration is achieved by an indirect ELISA format. In essence, PEA-15 (Phospho-Ser116) is captured by PEA-15 (Phospho-Ser116)-specific primary (1Ãƒ¸) antibodies while the HRP-conjugated secondary (2Ãƒ¸) antibodies bind the Fc region of the 1Ãƒ¸ antibody. Through this binding, the HRP enzyme conjugated to the 2Ãƒ¸ 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: 8682, UniProt ID: Q15121, OMIM: 603434, Unigene: Hs.517216
Gene Symbol:	PEA15
Sub Type:	Phospho

UniProt Protein Function:	PEA-15: an ubiquitously expressed anti-apoptotic protein. Inhibits both Fas- and Tnfrsf1a/Tnfr1-mediated caspase-8 activity and apoptosis. Interacts with CASP8/FLICE and FADD. Regulates glucose transport by controlling both the content of GLUT1 glucose transporters on the plasma membrane and the insulin-dependent trafficking of GLUT4 from the cell interior to the surface. Most abundant in tissues such as heart, brain, muscle and adipose tissue which utilize glucose as an energy source. Lower expression in glucose-producing tissues. Higher levels of expression are found in tissues from individuals with type 2 diabetes than in controls.
UniProt Protein Details:	Protein type:Motility/polarity/chemotaxis; Apoptosis Chromosomal Location of Human Ortholog: 1q21.1 Cellular Component: cytosol; microtubule associated complex Molecular Function:protein binding Biological Process: activation of MAPK activity; activation of MAPKK activity; apoptosis; axon guidance; carbohydrate transport; DNA damage checkpoint; epidermal growth factor receptor signaling pathway; fibroblast growth factor receptor signaling pathway; innate immune response; insulin receptor signaling pathway; MAPKKK cascade; negative regulation of glucose import; nerve growth factor receptor signaling pathway; Ras protein signal transduction; response to morphine; small GTPase mediated signal transduction; transport; vascular endothelial growth factor receptor signaling pathway
NCBI Summary:	This gene encodes a death effector domain-containing protein that functions as a negative regulator of apoptosis. The encoded protein is an endogenous substrate for protein kinase C. This protein is also overexpressed in type 2 diabetes mellitus, where it may contribute to insulin resistance in glucose uptake. Alternative splicing results in multiple transcript variants. [provided by RefSeq, Jul 2014]
UniProt Code:	Q15121
NCBI GenInfo Identifier:	32470612
NCBI Gene ID:	8682
NCBI Accession:	Q15121.2
UniProt Secondary Accession:	Q15121,O00511, B1AKZ3,
UniProt Related Accession:	Q15121
Molecular Weight:	17,307 Da
NCBI Full Name:	Astrocytic phosphoprotein PEA-15
NCBI Synonym Full Names:	phosphoprotein enriched in astrocytes 15
NCBI Official Symbol:	PEA15
NCBI Official Synonym Symbols:	PED; MAT1; HMAT1; MAT1H; PEA-15; HUMMAT1H
NCBI Protein Information:	astrocytic phosphoprotein PEA-15
UniProt Protein Name:	Astrocytic phosphoprotein PEA-15
UniProt Synonym Protein Names:	15 kDa phosphoprotein enriched in astrocytes; Phosphoprotein enriched in diabetes; PED
Protein Family:	Astrocytic phosphoprotein
UniProt Gene Name:	PEA15
UniProt Entry Name:	PEA15_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-PEA-15 (Phospho-Ser116) Antibody, Anti-PEA-15 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.)