CSFR (Phospho-Tyr561) Fluorometric Cell-Based ELISA Kit

SKU:
FBCAB00049
Product type:
ELISA Kit
ELISA Type:
Cell Based
€599

Description

Technical Manual

CSFR (Phospho-Tyr561) Cell-Based ELISA Kit

The CSFR (Phospho-Tyr561) Cell-Based ELISA Kit is a convenient, lysate- free, high throughput and sensitive assay kit that can monitor CSFR phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated CSFR in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on CSFR phosphorylation.

How does our CSFR (Phospho-Tyr561) Fluorometric Cell-Based ELISA Kit work?

Qualitative determination of CSFR (Phospho-Tyr561) concentration is achieved by an indirect ELISA format. In essence, CSFR (Phospho-Tyr561) is captured by CSFR (Phospho-Tyr561)-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 CSFR (Phospho-Tyr561) is also provided for normalization purposes. The RFU values obtained for non-phosphorylated CSFR can be used to normalize the RFU value for phosphorylated CSFR.

CSFR (Phospho-Tyr561) Fluorometric Cell-Based ELISA Kit -Information

Product Name:CSFR (Phospho-Tyr561) Fluorometric Cell-Based ELISA Kit
Product Code/SKU:FBCAB00049
Description:The CSFR (Phospho-Tyr561) Fluorometric Cell-Based Phospho ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can monitor CSFR (Phospho-Tyr561) protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated CSFR (Phospho-Tyr561) in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals, or activators have on CSF1R 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: 1436, UniProt ID: P07333, OMIM #: 164770, Unigene #: Hs.586219/Hs.654394
Format:Two 96-Well Plates
NCBI Gene Symbol:CSF1R
Sub Type:Phospho
Target Name:Phospho-CSFR (Tyr561)

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 CSFR (Phospho-Tyr561) detection. Add 50 µl of Primary Antibody Mixture NP to the corresponding wells for total CSFR 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.

CSFR (Phospho-Tyr561) - Protein Information

UniProt Protein Function:CSFR: an oncogenic tyrosine kinase receptor for CSF-1 (M-CSF). Drives growth and development of monocytes. Binding of CSF-1 induces receptor dimerization, activation and autophosphorylation of cytoplasmic tyrosine residues used as docking sites for SH2-containing signaling proteins. There are at least five major tyrosine autophosphorylation sites. Two point mutations seen in 10-20% of patients with acute myeloid leukemia, chronic myelomonocytic leukemia or myelodysplasia. One mutation appears to be both somatic and germline, and disrupts Cbl binding and receptor turnover. v-fms lacks the Cbl binding site and causes feline leukemia. Mutations may also develop after chemotherapy for lymphoma. A distinct point mutation was found in some cases of hepatocellular carcinoma and related to increased expression, and another mutation was found in 2 of 40 patients with idiopathic myelofibrosis. Expression is elevated in breast tumors and cell lines, and expression in xenografts and transgenic mice has been correlated with xenograft growth and breast cancer development. Inhibitors: Ki-20227 and other Kit/PDGFR inhibitors.
UniProt Protein Details:

Protein type:Kinase, protein; Membrane protein, integral; EC 2.7.10.1; Protein kinase, tyrosine (receptor); Oncoprotein; Protein kinase, TK; TK group; PDGFR family

Chromosomal Location of Human Ortholog: 5q32

Cellular Component: cell surface; integral to plasma membrane; intracellular; plasma membrane

Molecular Function:ATP binding; cytokine binding; macrophage colony stimulating factor receptor activity; protein homodimerization activity; protein phosphatase binding

Biological Process: axon guidance; cell proliferation; cytokine and chemokine mediated signaling pathway; forebrain neuron differentiation; hemopoiesis; inflammatory response; innate immune response; intercellular junction maintenance; macrophage differentiation; monocyte differentiation; multicellular organismal development; negative regulation of apoptosis; negative regulation of cell proliferation; olfactory bulb development; osteoclast differentiation; peptidyl-tyrosine phosphorylation; phosphatidylinositol metabolic process; phosphoinositide-mediated signaling; positive regulation of cell migration; positive regulation of cell proliferation; positive regulation of osteoclast differentiation; positive regulation of protein amino acid phosphorylation; positive regulation of tyrosine phosphorylation of Stat3 protein; protein amino acid autophosphorylation; regulation of bone resorption; regulation of cell shape; ruffle organization and biogenesis; signal transduction; skeletal muscle development; transmembrane receptor protein tyrosine kinase signaling pathway

Disease: Leukoencephalopathy, Diffuse Hereditary, With Spheroids

NCBI Summary:The protein encoded by this gene is the receptor for colony stimulating factor 1, a cytokine which controls the production, differentiation, and function of macrophages. This receptor mediates most if not all of the biological effects of this cytokine. Ligand binding activates the receptor kinase through a process of oligomerization and transphosphorylation. The encoded protein is a tyrosine kinase transmembrane receptor and member of the CSF1/PDGF receptor family of tyrosine-protein kinases. Mutations in this gene have been associated with a predisposition to myeloid malignancy. The first intron of this gene contains a transcriptionally inactive ribosomal protein L7 processed pseudogene oriented in the opposite direction. Alternative splicing results in multiple transcript variants. [provided by RefSeq, Dec 2013]
UniProt Code:P07333
NCBI GenInfo Identifier:547770
NCBI Gene ID:1436
NCBI Accession:P07333.2
UniProt Secondary Accession:P07333,Q6LDW5, Q6LDY4, Q86VW7, B5A955, D3DQG2,
UniProt Related Accession:P07333
Molecular Weight:33,248 Da
NCBI Full Name:Macrophage colony-stimulating factor 1 receptor
NCBI Synonym Full Names:colony stimulating factor 1 receptor
NCBI Official Symbol:CSF1R  
NCBI Official Synonym Symbols:FMS; CSFR; FIM2; HDLS; C-FMS; CD115; CSF-1R; M-CSF-R  
NCBI Protein Information:macrophage colony-stimulating factor 1 receptor
UniProt Protein Name:Macrophage colony-stimulating factor 1 receptor
UniProt Synonym Protein Names:CSF-1 receptor (EC:2.7.10.1); CSF-1-R; CSF-1R; M-CSF-R; Proto-oncogene c-Fms; CD_antigen: CD115
Protein Family:Macrophage colony-stimulating factor 1 receptor
UniProt Gene Name:CSF1R  
UniProt Entry Name:CSF1R_HUMAN