null

CrkII (Phospho-Tyr221) Fluorometric Cell-Based ELISA Kit

SKU:
FBCAB00011
Product Type:
ELISA Kit
ELISA Type:
Cell Based Phospho Specific
Reactivity:
Human
Mouse
Rat
Detection Method:
Fluorometric
$719
Frequently bought together:

Description

Technical Manual

CrkII (Phospho-Tyr221) Cell-Based ELISA Kit

The CrkII (Phospho-Tyr221) Cell-Based ELISA Kit is a convenient, lysate- free, high throughput and sensitive assay kit that can monitor CrkII phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated CrkII in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on CrkII phosphorylation.

How does our CrkII (Phospho-Tyr221) Fluorometric Cell-Based ELISA Kit work?

Qualitative determination of CrkII (Phospho-Tyr221) concentration is achieved by an indirect ELISA format. In essence, CrkII (Phospho-Tyr221) is captured by CrkII (Phospho-Tyr221)-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 CrkII (Phospho-Tyr221) is also provided for normalization purposes. The RFU values obtained for non-phosphorylated CrkII can be used to normalize the RFU value for phosphorylated CrkII.

CrkII (Phospho-Tyr221) Fluorometric Cell-Based ELISA Kit -Information

Product Name:CrkII (Phospho-Tyr221) Fluorometric Cell-Based ELISA Kit
Product Code/SKU:FBCAB00011
Description:The CrkII (Phospho-Tyr221) Fluorometric Cell-Based Phospho ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can monitor CrkII (Phospho-Tyr221) protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated CrkII (Phospho-Tyr221) in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals, or activators have on CRK 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: 1398, UniProt ID: P46108, OMIM #: 164762, Unigene #: Hs.638121
Format:Two 96-Well Plates
NCBI Gene Symbol:CRK
Sub Type:Phospho
Target Name:Phospho-CrkII (Tyr221)

Kit Principle

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

Kit components

Quantity

96-Well Black Cell CultureClear-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 Antibody110 µl
Dye-1 Conjugated Anti-Rabbit IgG Antibody6 ml
Dye-2 Conjugated Anti-Mouse IgG Antibody6 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:

  • 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 CrkII (Phospho-Tyr221) detection. Add 50 µl of Primary Antibody Mixture NP to the corresponding wells for total CrkII 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.

CrkII (Phospho-Tyr221) - Protein Information

UniProt Protein Function:CRK: an adaptor protein with an SH2-SH3-SH3 domain structure. Recruits cytoplasmic proteins through SH2-phospho-tyrosine interaction. Phosphorylated by Abl, IGF-IR and EGFR. Phosphorylation induces a change in intramolecular folding and SH2 interactions, causing its rapid dissociation from the tyrosine kinase complex.
UniProt Protein Details:

Protein type:Motility/polarity/chemotaxis; Adaptor/scaffold; Oncoprotein

Chromosomal Location of Human Ortholog: 17p13.3

Cellular Component: cytoplasm; plasma membrane; cytosol; nucleus; actin cytoskeleton

Molecular Function:protein phosphorylated amino acid binding; protein binding; ephrin receptor binding; SH3/SH2 adaptor activity; SH2 domain binding

Biological Process: regulation of transcription from RNA polymerase II promoter; regulation of Rac protein signal transduction; platelet activation; activation of MAPKK activity; nerve growth factor receptor signaling pathway; regulation of actin cytoskeleton organization and biogenesis; insulin receptor signaling pathway; ephrin receptor signaling pathway; innate immune response; vascular endothelial growth factor receptor signaling pathway; blood coagulation

NCBI Summary:This gene encodes a member of an adapter protein family that binds to several tyrosine-phosphorylated proteins. The product of this gene has several SH2 and SH3 domains (src-homology domains) and is involved in several signaling pathways, recruiting cytoplasmic proteins in the vicinity of tyrosine kinase through SH2-phosphotyrosine interaction. The N-terminal SH2 domain of this protein functions as a positive regulator of transformation whereas the C-terminal SH3 domain functions as a negative regulator of transformation. Two alternative transcripts encoding different isoforms with distinct biological activity have been described. [provided by RefSeq, Jul 2008]
UniProt Code:P46108
NCBI GenInfo Identifier:158939322
NCBI Gene ID:1398
NCBI Accession:P46108.2
UniProt Secondary Accession:P46108,Q96GA9, Q96HJ0, A8MWE8, B0LPE8, D3DTH6,
UniProt Related Accession:P46108
Molecular Weight:304
NCBI Full Name:Adapter molecule crk
NCBI Synonym Full Names:v-crk avian sarcoma virus CT10 oncogene homolog
NCBI Official Symbol:CRK  
NCBI Official Synonym Symbols:p38; CRKII  
NCBI Protein Information:adapter molecule crk; proto-oncogene c-Crk
UniProt Protein Name:Adapter molecule crk
UniProt Synonym Protein Names:Proto-oncogene c-Crk; p38
Protein Family:Crk-like protein
UniProt Gene Name:CRK  
UniProt Entry Name:CRK_HUMAN

Related Products