p56 Dok-2 (Phospho-Tyr299) Colorimetric Cell-Based ELISA Kit

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SKU:
CBCAB01516
Product Type:
ELISA Kit
ELISA Type:
Cell Based Phospho Specific
Research Area:
Immunology
Reactivity:
Human
Reactivity:
Mouse
Detection Method:
Colorimetric

Description

system_update_altDatasheet
Product Name:p56 Dok-2 (Phospho-Tyr299) Colorimetric Cell-Based ELISA
Product Code:CBCAB01516
ELISA Type:Cell-Based
Target:p56 Dok-2 (Phospho-Tyr299)
Reactivity:Human, Mouse
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nm
Format:2 x 96-Well Microplates

The p56 Dok-2 (Phospho-Tyr299) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect p56 Dok-2 protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated p56 Dok-2 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on p56 Dok-2 phosphorylation.

Qualitative determination of p56 Dok-2 (Phospho-Tyr299) concentration is achieved by an indirect ELISA format. In essence, p56 Dok-2 (Phospho-Tyr299) is captured by p56 Dok-2 (Phospho-Tyr299)-specific primary (1ø) antibodies while the HRP-conjugated secondary (2ø) antibodies bind the Fc region of the 1ø antibody. Through this binding, the HRP enzyme conjugated to the 2ø 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: 9046, UniProt ID: O60496, OMIM: 604997, Unigene: Hs.71215
Gene Symbol:DOK2
Sub Type:Phospho
UniProt Protein Function:DOK2: a member of the p62dok family of proteins that interact with receptor tyrosine kinases and mediate particular biological responses. Constitutively tyrosine phosphorylated in hematopoietic progenitors isolated from chronic myelogenous leukemia (CML) patients in the chronic phase. It may be a critical substrate for p210(bcr/Abl), a chimeric protein whose presence is associated with CML. Contains a pleckstrin homology domain at the amino terminus, 13 potential tyrosine phosphorylation sites, and six PXXP SH3 recognition motifs. Binds p120 (RasGAP) from CML cells.
UniProt Protein Details:

Protein type:Adaptor/scaffold

Chromosomal Location of Human Ortholog: 8p21.3

Cellular Component: cytosol

Molecular Function:insulin receptor binding; protein binding; receptor signaling protein activity; transmembrane receptor protein tyrosine kinase adaptor protein activity

Biological Process: blood coagulation; cell surface receptor linked signal transduction; leukocyte migration; Ras protein signal transduction; signal transduction; transmembrane receptor protein tyrosine kinase signaling pathway

NCBI Summary:The protein encoded by this gene is constitutively tyrosine phosphorylated in hematopoietic progenitors isolated from chronic myelogenous leukemia (CML) patients in the chronic phase. It may be a critical substrate for p210(bcr/abl), a chimeric protein whose presence is associated with CML. This encoded protein binds p120 (RasGAP) from CML cells. [provided by RefSeq, Jul 2008]
UniProt Code:O60496
NCBI GenInfo Identifier:143220234
NCBI Gene ID:9046
NCBI Accession:O60496.2
UniProt Secondary Accession:O60496,Q8N5A4,
UniProt Related Accession:O60496
Molecular Weight:45,379 Da
NCBI Full Name:Docking protein 2
NCBI Synonym Full Names:docking protein 2
NCBI Official Symbol:DOK2  
NCBI Official Synonym Symbols:p56DOK; p56dok-2  
NCBI Protein Information:docking protein 2
UniProt Protein Name:Docking protein 2
UniProt Synonym Protein Names:Downstream of tyrosine kinase 2; p56(dok-2)
Protein Family:Docking protein
UniProt Gene Name:DOK2  
UniProt Entry Name:DOK2_HUMAN
ComponentQuantity
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 µL
100x Anti-Target Antibody60 µL
Anti-GAPDH Antibody60 µL
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

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.

StepProcedure
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-p56 Dok-2 (Phospho-Tyr299) Antibody, Anti-p56 Dok-2 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.)

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