ZAP-70 (Phospho-Tyr315)Colorimetric Cell-Based ELISA Kit (CBCAB00057)

Product Name:	ZAP-70 (Phospho-Tyr315)Colorimetric Cell-Based ELISA Kit
Product Code:	CBCAB00057
ELISA Type:	Cell-Based
Target:	ZAP-70 (Phospho-Tyr315)
Reactivity:	Human, Mouse
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nm
Format:	2 x 96-Well Microplates

The ZAP-70 (Phospho-Tyr315) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect ZAP-70 protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated ZAP-70 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on ZAP-70 phosphorylation.

Qualitative determination of ZAP-70 (Phospho-Tyr315) concentration is achieved by an indirect ELISA format. In essence, ZAP-70 (Phospho-Tyr315) is captured by ZAP-70 (Phospho-Tyr315)-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: 7535, UniProt ID: P43403, OMIM: 176947, Unigene: Hs.234569
Gene Symbol:	ZAP70
Sub Type:	Phospho

UniProt Protein Function:	ZAP70: a tyrosine kinase of the Syk family. Associates with the T-cell antigen receptor zeta-chain after TCR stimulation. Phosphorylated by Src-family kinases following antigen receptor activation. Plays a role in lymphocyte activation.
UniProt Protein Details:	Protein type:Kinase, protein; Protein kinase, TK; EC 2.7.10.2; Protein kinase, tyrosine (non-receptor); TK group; Syk family Chromosomal Location of Human Ortholog: 2q12 Cellular Component: cytoplasm; cytosol; extrinsic to internal side of plasma membrane; immunological synapse; intercellular junction; lipid raft; plasma membrane; T cell receptor complex Molecular Function:ATP binding; non-membrane spanning protein tyrosine kinase activity; phosphotyrosine binding; protein binding; protein-tyrosine kinase activity; receptor binding Biological Process: adaptive immune response; B cell activation; B cell receptor signaling pathway; beta selection; immune response; innate immune response; macrophage activation during immune response; negative thymic T cell selection; neutrophil activation during immune response; peptidyl-tyrosine phosphorylation; positive regulation of alpha-beta T cell differentiation; positive regulation of alpha-beta T cell proliferation; positive regulation of B cell differentiation; positive regulation of calcium-mediated signaling; positive regulation of cell adhesion mediated by integrin; positive regulation of mast cell degranulation; positive regulation of T cell differentiation; positive thymic T cell selection; protein amino acid phosphorylation; T cell activation; T cell differentiation; T cell receptor signaling pathway; transmembrane receptor protein tyrosine kinase signaling pathway Disease: Selective T-cell Defect
NCBI Summary:	This gene encodes an enzyme belonging to the protein tyrosine kinase family, and it plays a role in T-cell development and lymphocyte activation. This enzyme, which is phosphorylated on tyrosine residues upon T-cell antigen receptor (TCR) stimulation, functions in the initial step of TCR-mediated signal transduction in combination with the Src family kinases, Lck and Fyn. This enzyme is also essential for thymocyte development. Mutations in this gene cause selective T-cell defect, a severe combined immunodeficiency disease characterized by a selective absence of CD8-positive T-cells. Two transcript variants that encode different isoforms have been found for this gene. [provided by RefSeq, Jul 2008]
UniProt Code:	P43403
NCBI GenInfo Identifier:	1177044
NCBI Gene ID:	7535
NCBI Accession:	P43403.1
UniProt Secondary Accession:	P43403,Q6PIA4, Q8IXD6, Q9UBS6, A6NFP4,
UniProt Related Accession:	P43403
Molecular Weight:	55,873 Da
NCBI Full Name:	Tyrosine-protein kinase ZAP-70
NCBI Synonym Full Names:	zeta chain of T cell receptor associated protein kinase 70kDa
NCBI Official Symbol:	ZAP70
NCBI Official Synonym Symbols:	SRK; STD; TZK; STCD; ZAP-70
NCBI Protein Information:	tyrosine-protein kinase ZAP-70
UniProt Protein Name:	Tyrosine-protein kinase ZAP-70
UniProt Synonym Protein Names:	70 kDa zeta-chain associated protein; Syk-related tyrosine kinase
Protein Family:	Tyrosine-protein kinase
UniProt Gene Name:	ZAP70
UniProt Entry Name:	ZAP70_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-ZAP-70 (Phospho-Tyr315) Antibody, Anti-ZAP-70 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.)