null

CXCR4 (Phospho-Ser339) Fluorometric Cell-Based ELISA Kit

SKU:
FBCAB00050
Product type:
ELISA Kit
ELISA Type:
Cell Based
€599

Description

Technical Manual

CXCR4 (Phospho-Ser339) Cell-Based ELISA Kit

The CXCR4 (Phospho-Ser339) Cell-Based ELISA Kit is a convenient, lysate- free, high throughput and sensitive assay kit that can monitor CXCR4 phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated CXCR4 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on CXCR4 phosphorylation.

How does our CXCR4 (Phospho-Ser339) Fluorometric Cell-Based ELISA Kit work?

Qualitative determination of CXCR4 (Phospho-Ser339) concentration is achieved by an indirect ELISA format. In essence, CXCR4 (Phospho-Ser339) is captured by CXCR4 (Phospho-Ser339)-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 CXCR4 (Phospho-Ser339) is also provided for normalization purposes. The RFU values obtained for non-phosphorylated CXCR4 can be used to normalize the RFU value for phosphorylated CXCR4.

CXCR4 (Phospho-Ser339) Fluorometric Cell-Based ELISA Kit -Information

Product Name:CXCR4 (Phospho-Ser339) Fluorometric Cell-Based ELISA Kit
Product Code/SKU:FBCAB00050
Description:The CXCR4 (Phospho-Ser339) Fluorometric Cell-Based Phospho ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can monitor CXCR4 (Phospho-Ser339) protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated CXCR4 (Phospho-Ser339) in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals, or activators have on CXCR4 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: 7852, UniProt ID: P61073, OMIM #: 162643/193670, Unigene #: Hs.593413
Format:Two 96-Well Plates
NCBI Gene Symbol:CXCR4
Sub Type:Phospho
Target Name:Phospho-CXCR4 (Ser339)

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 CXCR4 (Phospho-Ser339) detection. Add 50 µl of Primary Antibody Mixture NP to the corresponding wells for total CXCR4 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.

CXCR4 (Phospho-Ser339) - Protein Information

UniProt Protein Function:CXCR4: Receptor for the C-X-C chemokine CXCL12/SDF-1 that transduces a signal by increasing intracellular calcium ion levels and enhancing MAPK1/MAPK3 activation. Acts as a receptor for extracellular ubiquitin; leading to enhanced intracellular calcium ions and reduced cellular cAMP levels. Involved in hematopoiesis and in cardiac ventricular septum formation. Also plays an essential role in vascularization of the gastrointestinal tract, probably by regulating vascular branching and/or remodeling processes in endothelial cells. Involved in cerebellar development. In the CNS, could mediate hippocampal-neuron survival. Acts as a coreceptor (CD4 being the primary receptor) for HIV-1 X4 isolates and as a primary receptor for some HIV-2 isolates. Promotes Env-mediated fusion of the virus. Monomer. Can form dimers. Interacts with CD164. Interacts with HIV-1 surface protein gp120 and Tat. Interacts with ARRB2; the interaction is dependent on the C-terminal phosphorylation of CXCR4 and allows activation of MAPK1 and MAPK3. Interacts with ARRC; the interaction is dependent on the C-terminal phosphorylation of CXCR4 and modulates calcium mobilization. Interacts (via the cytoplasmic C-terminal) with ITCH (via the WW domains I and II); the interaction, enhanced by CXCL12, ubiquitinates CXCR4 and leads to its degradation. Interacts with extracellular ubiquitin. Interacts with human cytomegalovirus/HHV- 5 protein UL78. Expressed in numerous tissues, such as peripheral blood leukocytes, spleen, thymus, spinal cord, heart, placenta, lung, liver, skeletal muscle, kidney, pancreas, cerebellum, cerebral cortex and medulla (in microglia as well as in astrocytes), brain microvascular, coronary artery and umbilical cord endothelial cells. Isoform 1 is predominant in all tissues tested. Belongs to the G-protein coupled receptor 1 family. 2 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:

Protein type:Membrane protein, integral; Receptor, GPCR; GPCR, family 1; Membrane protein, multi-pass

Chromosomal Location of Human Ortholog: 2q21

Cellular Component: cell surface; lysosome; leading edge; early endosome; cytoplasmic membrane-bound vesicle; integral to membrane; intercellular junction; growth cone; late endosome; cytoplasm; plasma membrane; cytoplasmic vesicle; external side of plasma membrane

Molecular Function:G-protein coupled receptor activity; viral receptor activity; ubiquitin binding; protein binding; cytokine binding; ubiquitin protein ligase binding; myosin light chain binding; coreceptor activity; actin binding; C-X-C chemokine receptor activity

Biological Process: entry of virus into host cell; regulation of chemotaxis; viral reproduction; activation of MAPK activity; apoptosis; neuron migration; motor axon guidance; regulation of cell migration; germ cell development; T cell proliferation; elevation of cytosolic calcium ion concentration; germ cell migration; dendritic cell chemotaxis; positive regulation of oligodendrocyte differentiation; inflammatory response; neutrophil activation; response to virus; calcium-mediated signaling; patterning of blood vessels; G-protein coupled receptor protein signaling pathway; ameboidal cell migration; myelin maintenance; response to hypoxia; entry into host cell; brain development

Disease: Whim Syndrome

NCBI Summary:This gene encodes a CXC chemokine receptor specific for stromal cell-derived factor-1. The protein has 7 transmembrane regions and is located on the cell surface. It acts with the CD4 protein to support HIV entry into cells and is also highly expressed in breast cancer cells. Mutations in this gene have been associated with WHIM (warts, hypogammaglobulinemia, infections, and myelokathexis) syndrome. Alternate transcriptional splice variants, encoding different isoforms, have been characterized. [provided by RefSeq, Jul 2008]
UniProt Code:P61073
NCBI GenInfo Identifier:46577576
NCBI Gene ID:7852
NCBI Accession:P61073.1
UniProt Secondary Accession:P61073,O60835, P30991, P56438, Q53S69, Q9BXA0, Q9UKN2 B2R5N0,
UniProt Related Accession:P61073
Molecular Weight:40,221 Da
NCBI Full Name:C-X-C chemokine receptor type 4
NCBI Synonym Full Names:chemokine (C-X-C motif) receptor 4
NCBI Official Symbol:CXCR4  
NCBI Official Synonym Symbols:FB22; HM89; LAP3; LCR1; NPYR; WHIM; CD184; LAP-3; LESTR; NPY3R; NPYRL; HSY3RR; NPYY3R; D2S201E  
NCBI Protein Information:C-X-C chemokine receptor type 4; fusin; CD184 antigen; SDF-1 receptor; LPS-associated protein 3; neuropeptide Y receptor Y3; seven transmembrane helix receptor; stromal cell-derived factor 1 receptor; lipopolysaccharide-associated protein 3; seven-transmembrane-segment receptor, spleen; leukocyte-derived seven transmembrane domain receptor
UniProt Protein Name:C-X-C chemokine receptor type 4
UniProt Synonym Protein Names:FB22; Fusin; HM89; LCR1; Leukocyte-derived seven transmembrane domain receptor; LESTR; Lipopolysaccharide-associated protein 3; LAP-3; LPS-associated protein 3; NPYRL; Stromal cell-derived factor 1 receptor; SDF-1 receptor; CD_antigen: CD184
Protein Family:C-X-C chemokine receptor
UniProt Gene Name:CXCR4  
UniProt Entry Name:CXCR4_HUMAN