CCR5 (Phospho-Ser336)Colorimetric Cell-Based ELISA Kit (CBCAB01236)

Product Name:	CCR5 (Phospho-Ser336)Colorimetric Cell-Based ELISA Kit
Product Code:	CBCAB01236
ELISA Type:	Cell-Based
Target:	CCR5 (Phospho-Ser336)
Reactivity:	Human, Mouse, Rat
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nm
Format:	2 x 96-Well Microplates

The CCR5 (Phospho-Ser336) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect CCR5 protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated CCR5 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on CCR5 phosphorylation.

Qualitative determination of CCR5 (Phospho-Ser336) concentration is achieved by an indirect ELISA format. In essence, CCR5 (Phospho-Ser336) is captured by CCR5 (Phospho-Ser336)-specific primary (1Ãƒ¸) antibodies while the HRP-conjugated secondary (2Ãƒ¸) antibodies bind the Fc region of the 1Ãƒ¸ antibody. Through this binding, the HRP enzyme conjugated to the 2Ãƒ¸ 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: 1234/727797, UniProt ID: P51681, OMIM: 601373/609423/61037/612522, Unigene: Hs.536735
Gene Symbol:	CCR5
Sub Type:	Phospho

UniProt Protein Function:	CCR5: a 7-transmembrane G-linked receptor for a number of inflammatory C-C type chemokines including MIP-1-alpha, MIP-1-beta and RANTES. Transduces a signal by increasing the intracellular calcium ion level. May play a role in the control of granulocytic lineage proliferation or differentiation. Acts as a coreceptor (along with CD4) for HIV-1 R5 isolates. Interacts with PRAF2. Interacts with HIV-1 surface protein gp120. Efficient ligand binding to CCL3/MIP-1alpha and CCR4/MIP-1beta requires sulfation, O-glycosylation and sialic acid modifications. Glycosylation on S6 is required for efficient binding of CCL4. Interacts with ADRBK1. Interacts with ARRB1 and ARRB2. Variations in CCR5 are associated with resistance or susceptibility to immunodeficiency virus type 1 (resistance or susceptibility to HIV-1). Variations in CCR5 gene also influence the rate of progression to AIDS after infection. R60S variant, a naturally occurring mutation in a conserved residue in the first intracellular domain of CCR5, results in reduced amounts of the protein in the membrane and consequently may be associated with reduced susceptibility to infection by microbes that depend on these molecules as their receptors. Variations in CCR5 are associated with susceptibility to West Nile virus (WNV) infection
UniProt Protein Details:	Protein type:Receptor, GPCR; Motility/polarity/chemotaxis; Membrane protein, integral; Receptor, cytokine; Membrane protein, multi-pass; GPCR, family 1 Chromosomal Location of Human Ortholog: 3p21.31 Cellular Component: cell surface; integral to plasma membrane; cytoplasm; plasma membrane; endosome; external side of plasma membrane Molecular Function:protein binding; C-C chemokine receptor activity; chemokine receptor activity; coreceptor activity; C-C chemokine binding; actin binding; phosphoinositide phospholipase C activity Biological Process: viral reproduction; MAPKKK cascade; calcium-mediated signaling; release of sequestered calcium ion by sarcoplasmic reticulum into cytosol; chemotaxis; G-protein coupled receptor protein signaling pathway; elevation of cytosolic calcium ion concentration; cell surface receptor linked signal transduction; cell-cell signaling; calcium ion transport; dendritic cell chemotaxis; cellular defense response; immune response; entry into host cell; inflammatory response Disease: West Nile Virus, Susceptibility To; Diabetes Mellitus, Insulin-dependent, 22; Hepatitis C Virus, Susceptibility To
NCBI Summary:	This gene encodes a member of the beta chemokine receptor family, which is predicted to be a seven transmembrane protein similar to G protein-coupled receptors. This protein is expressed by T cells and macrophages, and is known to be an important co-receptor for macrophage-tropic virus, including HIV, to enter host cells. Defective alleles of this gene have been associated with the HIV infection resistance. The ligands of this receptor include monocyte chemoattractant protein 2 (MCP-2), macrophage inflammatory protein 1 alpha (MIP-1 alpha), macrophage inflammatory protein 1 beta (MIP-1 beta) and regulated on activation normal T expressed and secreted protein (RANTES). Expression of this gene was also detected in a promyeloblastic cell line, suggesting that this protein may play a role in granulocyte lineage proliferation and differentiation. This gene is located at the chemokine receptor gene cluster region. Two transcript variants encoding the same protein have been found for this gene. [provided by RefSeq, Jul 2008]
UniProt Code:	P51681
NCBI GenInfo Identifier:	1705896
NCBI Gene ID:	1234
NCBI Accession:	P51681.1
UniProt Secondary Accession:	P51681,O14692, O14693, O14695, O14696, O14697, O14698 O14699, O14700, O14701, O14702, O14703,
UniProt Related Accession:	P51681
Molecular Weight:	40,524 Da
NCBI Full Name:	C-C chemokine receptor type 5
NCBI Synonym Full Names:	chemokine (C-C motif) receptor 5 (gene/pseudogene)
NCBI Official Symbol:	CCR5
NCBI Official Synonym Symbols:	CKR5; CCR-5; CD195; CKR-5; CCCKR5; CMKBR5; IDDM22; CC-CKR-5
NCBI Protein Information:	C-C chemokine receptor type 5; chemr13; HIV-1 fusion coreceptor; chemokine receptor CCR5; C-C motif chemokine receptor 5 A159A
UniProt Protein Name:	C-C chemokine receptor type 5
UniProt Synonym Protein Names:	CHEMR13; HIV-1 fusion coreceptor; CD_antigen: CD195
Protein Family:	C-C chemokine receptor
UniProt Gene Name:	CCR5
UniProt Entry Name:	CCR5_HUMAN

Component	Quantity
96-Well Cell Culture Clear-Bottom Microplate	2 plates
10X TBS	24 mL
Quenching Buffer	24 mL
Blocking Buffer	50 mL
15X Wash Buffer	50 mL
Primary Antibody Diluent	12 mL
100x Anti-Phospho Target Antibody	60 µL
100x Anti-Target Antibody	60 µL
Anti-GAPDH Antibody	60 µL
HRP-Conjugated Anti-Rabbit IgG Antibody	12 mL
HRP-Conjugated Anti-Mouse IgG Antibody	12 mL
SDS Solution	12 mL
Stop Solution	24 mL
Ready-to-Use Substrate	12 mL
Crystal Violet Solution	12 mL
Adhesive Plate Seals	2 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.

Step	Procedure
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-CCR5 (Phospho-Ser336) Antibody, Anti-CCR5 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.)