Mouse Ccr5 ELISA Kit (MOEB1884)- High Sensitivity

Product Name:	Mouse C-C chemokine receptor type 5 (Ccr5) ELISA Kit
Product Code:	MOEB1884
Alias:	C-C chemokine receptor type 5, C-C CKR-5, CC-CKR-5, CCR-5, MIP-1 alpha receptor, Ccr5, Cmkbr5, CD195
Uniprot:	P51682
Reactivity:	Mouse
Range:	0.156-10 ng/mL
Detection Method:	Sandwich
Size:	96 Assay
Storage:	Please see kit components below for exact storage details
Note:	For research use only

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:Membrane protein, multi-pass; Receptor, cytokine; Motility/polarity/chemotaxis; GPCR, family 1; Membrane protein, integral; Receptor, GPCR Cellular Component: cell surface; membrane; cytoplasm; integral to membrane; plasma membrane; cytosol; endosome; external side of plasma membrane Molecular Function:G-protein coupled receptor activity; signal transducer activity; C-C chemokine receptor activity; chemokine receptor activity; C-C chemokine binding; actin binding; glycoprotein binding; protein kinase binding Biological Process: calcium-mediated signaling; positive regulation of interleukin-6 production; defense response; positive regulation of interleukin-1 beta secretion; negative regulation of axon extension; chemotaxis; release of sequestered calcium ion by sarcoplasmic reticulum into cytosol; positive regulation of tumor necrosis factor production; signal transduction; positive regulation of apoptosis by virus; G-protein coupled receptor protein signaling pathway; elevation of cytosolic calcium ion concentration; cell-cell signaling; calcium ion transport; positive regulation of fever; positive regulation of cell-cell adhesion; immune response; positive regulation of neuron differentiation; inflammatory response; negative regulation of cell migration; positive regulation of inflammatory response
UniProt Code:	P51682
NCBI GenInfo Identifier:	341940545
NCBI Gene ID:	12774
NCBI Accession:	P51682.3
UniProt Secondary Accession:	P51682,O35313, O35891, P97308, P97405, Q3ZAZ8, Q61867
UniProt Related Accession:	P51682
Molecular Weight:	40,785 Da
NCBI Full Name:	C-C chemokine receptor type 5
NCBI Synonym Full Names:	chemokine (C-C motif) receptor 5
NCBI Official Symbol:	Ccr5
NCBI Official Synonym Symbols:	AM4-7; CD195; Cmkbr5
NCBI Protein Information:	C-C chemokine receptor type 5; CCR-5; CC-CKR-5; C-C CKR-5; MIP-1 alpha receptor; chemokine (C-C) receptor 5
UniProt Protein Name:	C-C chemokine receptor type 5
UniProt Synonym Protein Names:	MIP-1 alpha receptor; CD_antigen: CD195
Protein Family:	C-C chemokine receptor
UniProt Gene Name:	Ccr5
UniProt Entry Name:	CCR5_MOUSE

Component	Quantity (96 Assays)	Storage
ELISA Microplate (Dismountable)	8×12 strips	-20°C
Lyophilized Standard	2	-20°C
Sample Diluent	20ml	-20°C
Assay Diluent A	10mL	-20°C
Assay Diluent B	10mL	-20°C
Detection Reagent A	120µL	-20°C
Detection Reagent B	120µL	-20°C
Wash Buffer	30mL	4°C
Substrate	10mL	4°C
Stop Solution	10mL	4°C
Plate Sealer	5	-

Other materials and equipment required:

Microplate reader with 450 nm wavelength filter
Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
Incubator
Deionized or distilled water
Absorbent paper
Buffer resevoir

*Note: The below protocol is a sample protocol. Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

Allow all reagents to reach room temperature (Please do not dissolve the reagents at 37°C directly). All the reagents should be mixed thoroughly by gently swirling before pipetting. Avoid foaming. Keep appropriate numbers of strips for 1 experiment and remove extra strips from microtiter plate. Removed strips should be resealed and stored at -20°C until the kits expiry date. Prepare all reagents, working standards and samples as directed in the previous sections. Please predict the concentration before assaying. If values for these are not within the range of the standard curve, users must determine the optimal sample dilutions for their experiments. We recommend running all samples in duplicate.

Step
1.	Add Sample: Add 100µL of Standard, Blank, or Sample per well. The blank well is added with Sample diluent. Solutions are added to the bottom of micro ELISA plate well, avoid inside wall touching and foaming as possible. Mix it gently. Cover the plate with sealer we provided. Incubate for 120 minutes at 37°C.
2.	Remove the liquid from each well, don't wash. Add 100µL of Detection Reagent A working solution to each well. Cover with the Plate sealer. Gently tap the plate to ensure thorough mixing. Incubate for 1 hour at 37°C. Note: if Detection Reagent A appears cloudy warm to room temperature until solution is uniform.
3.	Aspirate each well and wash, repeating the process three times. Wash by filling each well with Wash Buffer (approximately 400µL) (a squirt bottle, multi-channel pipette,manifold dispenser or automated washer are needed). Complete removal of liquid at each step is essential. After the last wash, completely remove remaining Wash Buffer by aspirating or decanting. Invert the plate and pat it against thick clean absorbent paper.
4.	Add 100µL of Detection Reagent B working solution to each well. Cover with the Plate sealer. Incubate for 60 minutes at 37°C.
5.	Repeat the wash process for five times as conducted in step 3.
6.	Add 90µL of Substrate Solution to each well. Cover with a new Plate sealer and incubate for 10-20 minutes at 37°C. Protect the plate from light. The reaction time can be shortened or extended according to the actual color change, but this should not exceed more than 30 minutes. When apparent gradient appears in standard wells, user should terminatethe reaction.
7.	Add 50µL of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing.
8.	Determine the optical density (OD value) of each well at once, using a micro-plate reader set to 450 nm. User should open the micro-plate reader in advance, preheat the instrument, and set the testing parameters.
9.	After experiment, store all reagents according to the specified storage temperature respectively until their expiry.

When carrying out an ELISA assay it is important to prepare your samples in order to achieve the best possible results. Below we have a list of procedures for the preparation of samples for different sample types.

Sample Type	Protocol
Serum	If using serum separator tubes, allow samples to clot for 30 minutes at room temperature. Centrifuge for 10 minutes at 1,000x g. Collect the serum fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. If serum separator tubes are not being used, allow samples to clot overnight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Remove serum and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.
Plasma	Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples at 4°C for 15 mins at 1000 × g within 30 mins of collection. Collect the plasma fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. Note: Over haemolysed samples are not suitable for use with this kit.
Urine & Cerebrospinal Fluid	Collect the urine (mid-stream) in a sterile container, centrifuge for 20 mins at 2000-3000 rpm. Remove supernatant and assay immediately. If any precipitation is detected, repeat the centrifugation step. A similar protocol can be used for cerebrospinal fluid.
Cell culture supernatant	Collect the cell culture media by pipette, followed by centrifugation at 4°C for 20 mins at 1500 rpm. Collect the clear supernatant and assay immediately.
Cell lysates	Solubilize cells in lysis buffer and allow to sit on ice for 30 minutes. Centrifuge tubes at 14,000 x g for 5 minutes to remove insoluble material. Aliquot the supernatant into a new tube and discard the remaining whole cell extract. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Tissue homogenates	The preparation of tissue homogenates will vary depending upon tissue type. Rinse tissue with 1X PBS to remove excess blood & homogenize in 20ml of 1X PBS (including protease inhibitors) and store overnight at ≤ -20°C. Two freeze-thaw cycles are required to break the cell membranes. To further disrupt the cell membranes you can sonicate the samples. Centrifuge homogenates for 5 mins at 5000xg. Remove the supernatant and assay immediately or aliquot and store at -20°C or -80°C.
Tissue lysates	Rinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with a tissue homogenizer in PBS. Add an equal volume of RIPA buffer containing protease inhibitors and lyse tissues at room temperature for 30 minutes with gentle agitation. Centrifuge to remove debris. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Breast Milk	Collect milk samples and centrifuge at 10,000 x g for 60 min at 4°C. Aliquot the supernatant and assay. For long term use, store samples at -80°C. Minimize freeze/thaw cycles.

ELISA

Mouse CCR5 / CD195 ELISA Kit