Src (Phospho-Tyr418) Fluorometric Cell-Based ELISA Kit

Description

Description

Src (Phospho-Tyr418) Cell-Based ELISA Kit

The Src (Phospho-Tyr418) Cell-Based ELISA Kit is a convenient, lysate- free, high throughput and sensitive assay kit that can monitor Src phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated Src in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on Src phosphorylation.

How does our Src (Phospho-Tyr418) Fluorometric Cell-Based ELISA Kit work?

Qualitative determination of Src (Phospho-Tyr418) concentration is achieved by an indirect ELISA format. In essence, Src (Phospho-Tyr418) is captured by Src (Phospho-Tyr418)-specific primary (1°) antibodies while Dye 1-conjugated and Dye 2-conjugated secondary (2°) antibodies bind the Fc region of the 1° antibody. Through this binding, the dye conjugated to the 2° antibody can emit light at a certain wavelength given proper excitation, hence allowing for a fluorometric detection method. 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 RFU values.
2.	An antibody against the nonphosphorylated counterpart of Src (Phospho-Tyr418) is also provided for normalization purposes. The RFU values obtained for non-phosphorylated Src can be used to normalize the RFU value for phosphorylated Src.

Src (Phospho-Tyr418) Fluorometric Cell-Based ELISA Kit -Information

Product Name:	Src (Phospho-Tyr418) Fluorometric Cell-Based ELISA Kit
Product Code/SKU:	FBCAB00007
Description:	The Src (Phospho-Tyr418) Fluorometric Cell-Based Phospho ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can monitor Src (Phospho-Tyr418) protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated Src (Phospho-Tyr418) in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals, or activators have on SRC phosphorylation.
Dynamic Range:	> 5000 Cells
Detection Method:	Fluorometric
Storage/Stability:	4°C/6 Months
Reactivity:	Human, Mouse, Rat
Assay Type:	Cell-Based ELISA
Database Links:	Gene ID: 6714, UniProt ID: P12931, OMIM #: 190090, Unigene #: Hs.195659
Format:	Two 96-Well Plates
NCBI Gene Symbol:	SRC
Sub Type:	Phospho
Target Name:	Phospho-Src (Tyr418)

Kit Principle

Figure: Schematic representation of Assay Genie Cell-Based Fluorometric ELISA principle

Kit components	Quantity
96-Well Black Cell CultureClear-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	110 µl
Dye-1 Conjugated Anti-Rabbit IgG Antibody	6 ml
Dye-2 Conjugated Anti-Mouse IgG Antibody	6 ml
Adhesive Plate Seals	2 seals

Additional equipment and materials required

The following materials and/or equipment are NOT provided in this kit but are necessary to successfully conduct the experiment:

Fluorescent plate reader with two channels at Ex/Em: 651/667 and 495/521
Micropipettes capable of measuring volumes from 1 µl to 1 ml
Deionized or sterile water (ddH2O)
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)

Kit Protocol

This is a summarized version of the kit protocol. Please view the technical manual of this kit for information on sample preparation, reagent preparation and plate lay out.

1.	Seed 200 µl of desired cell concentration in culture medium into each well of the 96-well plates. 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 3 minutes. 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 3 minutes each time.
9.	Dispense 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 3 minutes each time.
11.	Add 50 µl of Primary Antibody Mixture P to corresponding wells for Src (Phospho-Tyr418) detection. Add 50 µl of Primary Antibody Mixture NP to the corresponding wells for total Src detection. Cover the plate 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 3 minutes each time.
13.	Add 50 ul of Secondary Antibody Mixture to corresponding wells and incubate for 1.5 hours at room temperature in the dark.
14.	Wash 3 times with 200 µl of 1x Wash Buffer for 3 minutes each time.
15.	Read the plate(s) at Ex/Em: 651/667 (Dye 1) and 495/521 (Dye 2). Shield plates from direct light exposure.
16.	Wash 3 times with 200 µl of 1x Wash Buffer for 5 minutes each time.

Src (Phospho-Tyr418) - Protein Information

UniProt Protein Function:	Src: proto-oncogenic cytoplasmic tyrosine kinase of the SRC family. Highly expressed in certain fully differentiated cells such as neurons, platelets and macrophages. Phosphorylation of an activation loop tyrosine activates the enzyme; phosphorylation of a tyrosine in the C-terminus by Csk inhibits the enzyme. Two alternatively spliced isoforms have been described.
UniProt Protein Details:	Protein type:Oncoprotein; Protein kinase, tyrosine (non-receptor); Kinase, protein; EC 2.7.10.2; Protein kinase, TK; TK group; Src family Chromosomal Location of Human Ortholog: 20q12-q13 Cellular Component: neuron projection; mitochondrion; lysosome; postsynaptic density; actin filament; caveola; cytosol; extrinsic to internal side of plasma membrane; perinuclear region of cytoplasm; late endosome; mitochondrial inner membrane; cytoplasm; plasma membrane; nucleus Molecular Function:protein C-terminus binding; ephrin receptor binding; non-membrane spanning protein tyrosine kinase activity; phosphoprotein binding; insulin receptor binding; protein kinase activity; integrin binding; protein binding; enzyme binding; SH3/SH2 adaptor activity; protein kinase C binding; protein-tyrosine kinase activity; heme binding; estrogen receptor binding; SH2 domain binding; kinase activity; ATP binding; receptor binding; hormone receptor binding Biological Process: oogenesis; regulation of estrogen receptor signaling pathway; estrogen receptor signaling pathway; positive regulation of cyclin-dependent protein kinase activity; central nervous system development; progesterone receptor signaling pathway; nerve growth factor receptor signaling pathway; viral reproduction; regulation of cell cycle; positive regulation of apoptosis; positive regulation of transcription, DNA-dependent; uterus development; negative regulation of mitochondrial depolarization; negative regulation of protein homooligomerization; positive regulation of MAP kinase activity; cell-cell adhesion; transforming growth factor beta receptor signaling pathway; response to electrical stimulus; cell adhesion; bone resorption; response to drug; platelet activation; fibroblast growth factor receptor signaling pathway; activation of protein kinase B; response to virus; transcytosis; positive regulation of integrin activation; positive regulation of protein amino acid autophosphorylation; cellular response to insulin stimulus; response to mechanical stimulus; T cell costimulation; regulation of vascular permeability; negative regulation of transcription, DNA-dependent; leukocyte migration; negative regulation of apoptosis; axon guidance; peptidyl-tyrosine phosphorylation; protein amino acid autophosphorylation; platelet-derived growth factor receptor signaling pathway; negative regulation of caspase activity; signal transduction; positive regulation of smooth muscle cell migration; regulation of cell-cell adhesion; forebrain development; ephrin receptor signaling pathway; epidermal growth factor receptor signaling pathway; integrin-mediated signaling pathway; response to nutrient levels; regulation of bone resorption; negative regulation of focal adhesion formation; signal complex assembly; positive regulation of phosphoinositide 3-kinase activity; response to mineralocorticoid stimulus; cell cycle; regulation of cell proliferation; cell proliferation; positive regulation of protein kinase B signaling cascade; peptidyl-serine phosphorylation; response to hydrogen peroxide; regulation of protein binding; Ras protein signal transduction; stress fiber formation; innate immune response; response to acidity; positive regulation of insulin receptor signaling pathway; vascular endothelial growth factor receptor signaling pathway; blood coagulation; positive regulation of cytokine secretion
NCBI Summary:	This gene is highly similar to the v-src gene of Rous sarcoma virus. This proto-oncogene may play a role in the regulation of embryonic development and cell growth. The protein encoded by this gene is a tyrosine-protein kinase whose activity can be inhibited by phosphorylation by c-SRC kinase. Mutations in this gene could be involved in the malignant progression of colon cancer. Two transcript variants encoding the same protein have been found for this gene. [provided by RefSeq, Jul 2008]
UniProt Code:	P12931
NCBI GenInfo Identifier:	125711
NCBI Gene ID:	6714
NCBI Accession:	P12931.3
UniProt Secondary Accession:	P12931,Q76P87, Q86VB9, Q9H5A8, E1P5V4,
UniProt Related Accession:	P12931
Molecular Weight:	60kDa
NCBI Full Name:	Proto-oncogene tyrosine-protein kinase Src
NCBI Synonym Full Names:	SRC proto-oncogene, non-receptor tyrosine kinase
NCBI Official Symbol:	SRC
NCBI Official Synonym Symbols:	ASV; SRC1; c-SRC; p60-Src
NCBI Protein Information:	proto-oncogene tyrosine-protein kinase Src; proto-oncogene c-Src; tyrosine kinase pp60c-src; tyrosine-protein kinase SRC-1; protooncogene SRC, Rous sarcoma; v-src avian sarcoma (Schmidt-Ruppin A-2) viral oncogene homolog
UniProt Protein Name:	Proto-oncogene tyrosine-protein kinase Src
UniProt Synonym Protein Names:	Proto-oncogene c-Src; pp60c-src; p60-Src
Protein Family:	Proto-oncogene tyrosine-protein kinase
UniProt Gene Name:	SRC
UniProt Entry Name:	SRC_HUMAN