EFNB1 (Phospho-Tyr317) Colorimetric Cell-Based ELISA Kit

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SKU:
CBCAB01421
€599

Description

EFNB1 (Phospho-Tyr317) Cell-Based ELISA Kit

The EFNB1 (Phospho-Tyr317) Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can monitor EFNB1 (Phospho-Tyr317) protein expression profile in cells. The kit can be used for measuring the relative amounts of EFNB1 (Phospho-Tyr317) in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on EFNB1 phosphorylation.

How does our EFNB1 (Phospho-Tyr317) Colorimetric Cell-Based ELISA Kit work?

Qualitative determination of EFNB1 (Phospho-Tyr317) concentration is achieved by an indirect ELISA format. In essence, EFNB1 (Phospho-Tyr317) is captured by EFNB1 (Phospho-Tyr317) -specific primary (1°) antibodies while the HRP-conjugated secondary (2°) antibodies bind the Fc region of the 1° antibody. Through this binding, the HRP enzyme conjugated to the 2° 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.

EFNB1 (Phospho-Tyr317) Colorimetric Cell-Based ELISA Kit -Information

Product Name:EFNB1 (Phospho-Tyr317) Colorimetric Cell-Based ELISA Kit
Product Code/SKU:CBCAB01421
Description:The EFNB1 (Phospho-Tyr317) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect EFNB1 protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated EFNB1 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on EFNB1 phosphorylation.
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nm
Reactivity:Human, Mouse, Rat
Assay Type:Cell-Based
Format:2 x 96-Well Microplates
Database Links:Gene ID: 1947, UniProt ID: P98172, OMIM #: 300035/304110, Unigene #: Hs.144700
NCBI Gene Symbol:EFNB1
Sub Type:Phospho
Target Name:EFNB1 (Phospho-Tyr317)

Kit Principle

Figure: Schematic representation of ELISA Genie Cell-Based Colorimetric ELISA principle

Kit components

Quantity

96-Well Cell Culture Clear-Bottom Microplate2 plates
10X TBS24 ml
Quenching Buffer24 ml
Blocking Buffer50 ml
15X Wash Buffer50 ml
Primary Antibody Diluent12 ml
100x Anti-Phospho Target Antibody 60 ul
100x Anti-Target Antibody60 ul
Anti-GAPDH Antibody60 ul
HRP-Conjugated Anti-Rabbit IgG Antibody12 ml
HRP-Conjugated Anti-Mouse IgG Antibody12 ml
SDS Solution12 ml
Stop Solution24 ml
Ready-to-Use Substrate12 ml
Crystal Violet Solution12 ml
Adhesive Plate Seals2 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:

  • 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 ?l 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

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 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-EFNB1 (Phospho-Tyr317) Antibody, Anti-EFNB1 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.)

EFNB1 (Phospho-Tyr317) - Protein Information

UniProt Protein Function:EFNB1: a type I membrane protein of the ephrin family. A ligand of Eph-related receptor tyrosine kinases EphB1 and EphA1. Ephrins and ephrin receptors mediate numerous developmental processes, particularly in the nervous system. Ephrin-B1 may play a role in cell adhesion and functions in the development or maintenance of the nervous system. Binding to its receptor induces the collapse of commissural axons/growth cones in vitro. Induced by TNF-alpha. Expressed in brain, heart, placenta, lung, liver, skeletal muscle, kidney, and pancreas.
UniProt Protein Details:

Protein type:Membrane protein, integral; Ligand, receptor tyrosine kinase

Chromosomal Location of Human Ortholog: Xq12

Cellular Component: integral to plasma membrane; cytoplasm; plasma membrane; synapse; nucleus; lipid raft

Molecular Function:protein binding; ephrin receptor binding

Biological Process: axon guidance; cell-cell signaling; ephrin receptor signaling pathway; positive regulation of T cell proliferation; cell adhesion; embryonic pattern specification; neural crest cell migration

Disease: Craniofrontonasal Syndrome

NCBI Summary:The protein encoded by this gene is a type I membrane protein and a ligand of Eph-related receptor tyrosine kinases. It may play a role in cell adhesion and function in the development or maintenance of the nervous system. [provided by RefSeq, Jul 2008]
UniProt Code:P98172
NCBI GenInfo Identifier:1706668
NCBI Gene ID:1947
NCBI Accession:P98172.1
UniProt Secondary Accession:P98172,D3DVU0,
UniProt Related Accession:P98172
Molecular Weight:346
NCBI Full Name:Ephrin-B1
NCBI Synonym Full Names:ephrin-B1
NCBI Official Symbol:EFNB1  
NCBI Official Synonym Symbols:CFND; CFNS; EFB1; EFL3; EPLG2; Elk-L; LERK2  
NCBI Protein Information:ephrin-B1; EFL-3; LERK-2; ELK ligand; ligand of eph-related kinase 2; eph-related receptor tyrosine kinase ligand 2
UniProt Protein Name:Ephrin-B1
UniProt Synonym Protein Names:EFL-3; ELK ligand; ELK-L; EPH-related receptor tyrosine kinase ligand 2; LERK-2
Protein Family:Ephrin
UniProt Gene Name:EFNB1  
UniProt Entry Name:EFNB1_HUMAN
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