Human CCR6 ELISA Kit (HUEB2105)- High Sensitivity

Product Name:	Human C-C chemokine receptor type 6 (CCR6) ELISA Kit
SKU:	HUEB2105
Size:	96T
Target:	Human C-C chemokine receptor type 6 (CCR6)
Synonyms:	Chemokine receptor-like 3, DRY6, G-protein coupled receptor 29, GPR-CY4, LARC receptor, CKR-L3, GPRCY4, CD196, C-C CKR-6, CKRL3, CMKBR6, GPR29, STRL22
Assay Type:	Sandwich
Detection Method:	ELISA
Reactivity:	Human
Detection Range:	15.6-1000pg/mL
Sensitivity:	7.3pg/mL

Intra CV:

5.2%

Inter CV:

7.8%

Linearity:

Sample	1:2	1:4	1:8	1:16
Serum(N=5)	107-117%	92-103%	92-101%	100-112%
EDTA Plasma(N=5)	100-110%	97-107%	95-105%	81-90%
Heparin Plasma(N=5)	80-90%	87-97%	97-107%	90-99%

Recovery:

Sample Type	Average(%)	Recovery Range(%)
Serum	83	80-89
Plasma	85	80-91

Function:

Receptor for the C-C type chemokine CCL20 (PubMed:9169459). Binds to CCL20 and subsequently transduces a signal by increasing the intracellular calcium ion levels (PubMed:20068036). Although CCL20 is its major ligand it can also act as a receptor for non-chemokine ligands such as beta-defensins (PubMed:25585877). Binds to defensin DEFB1 leading to increase in intracellular calcium ions and cAMP levels. Its binding to DEFB1 is essential for the function of DEFB1 in regulating sperm motility and bactericidal activity (PubMed:25122636). Binds to defensins DEFB4 and DEFB4A/B and mediates their chemotactic effects (PubMed:20068036). The ligand-receptor pair CCL20-CCR6 is responsible for the chemotaxis of dendritic cells (DC), effector/ memory T-cells and B-cells and plays an important role at skin and mucosal surfaces under homeostatic and inflammatory conditions, as well as in pathology, including cancer and various autoimmune diseases. CCR6-mediated signals are essential for immune responses to microbes in the intestinal mucosa and in the modulation of inflammatory responses initiated by tissue insult and trauma (PubMed:21376174). CCR6 is essential for the recruitment of both the proinflammatory IL17 producing helper T-cells (Th17) and the regulatory T-cells (Treg) to sites of inflammation. Required for the normal migration of Th17 cells in Peyers-patches and other related tissue sites of the intestine and plays a role in regulating effector T-cell balance and distribution in inflamed intestine. Plays an important role in the coordination of early thymocyte precursor migration events important for normal subsequent thymocyte precursor development, but is not required for the formation of normal thymic natural regulatory T-cells (nTregs). Required for optimal differentiation of DN2 and DN3 thymocyte precursors. Essential for B-cell localization in the subepithelial dome of Peyers-patches and for efficient B-cell isotype switching to IgA in the Peyers-patches. Essential for appropriate anatomical distribution of memory B-cells in the spleen and for the secondary recall response of memory B-cells (By similarity). Positively regulates sperm motility and chemotaxis via its binding to CCL20 (PubMed:23765988).

Uniprot:	P51684
Sample Type:	Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:	Natural and recombinant human C-C chemokine receptor type 6
Research Area:	Immunology
Subcellular Location:	Cell membrane Multi-pass membrane protein Cell surface
Storage:	Please see kit components below for exact storage details
Note:	For research use only

UniProt Protein Function:	CCR6: Receptor for a C-C type chemokine. Binds to MIP-3- alpha/LARC and subsequently transduces a signal by increasing the intracellular calcium ions level. Belongs to the G-protein coupled receptor 1 family.
UniProt Protein Details:	Protein type:Receptor, GPCR; Membrane protein, integral; Motility/polarity/chemotaxis; GPCR, family 1; Membrane protein, multi-pass Chromosomal Location of Human Ortholog: 6q27 Cellular Component: cell surface; cytosol; integral to plasma membrane; plasma membrane Molecular Function:C-C chemokine receptor activity; chemokine receptor activity; receptor activity Biological Process: cell motility; cellular defense response; chemotaxis; dendritic cell chemotaxis; elevation of cytosolic calcium ion concentration; G-protein coupled receptor protein signaling pathway; humoral immune response; immune response; innate immune response; signal transduction
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. The gene is preferentially expressed by immature dendritic cells and memory T cells. The ligand of this receptor is macrophage inflammatory protein 3 alpha (MIP-3 alpha). This receptor has been shown to be important for B-lineage maturation and antigen-driven B-cell differentiation, and it may regulate the migration and recruitment of dentritic and T cells during inflammatory and immunological responses. Alternatively spliced transcript variants that encode the same protein have been described for this gene. [provided by RefSeq, Jul 2008]
UniProt Code:	P51684
NCBI GenInfo Identifier:	2851567
NCBI Gene ID:	1235
NCBI Accession:	P51684.2
UniProt Secondary Accession:	P51684,P78553, Q92846, E1P5C6,
UniProt Related Accession:	P51684
Molecular Weight:	42,494 Da
NCBI Full Name:	C-C chemokine receptor type 6
NCBI Synonym Full Names:	C-C motif chemokine receptor 6
NCBI Official Symbol:	CCR6
NCBI Official Synonym Symbols:	BN-1; DCR2; DRY6; CCR-6; CD196; CKRL3; GPR29; CKR-L3; CMKBR6; GPRCY4; STRL22; CC-CKR-6; C-C CKR-6
NCBI Protein Information:	C-C chemokine receptor type 6
UniProt Protein Name:	C-C chemokine receptor type 6
UniProt Synonym Protein Names:	Chemokine receptor-like 3; CKR-L3; DRY6; G-protein coupled receptor 29; GPR-CY4; GPRCY4; LARC receptor; CD_antigen: CD196
Protein Family:	C-C chemokine receptor
UniProt Gene Name:	CCR6
UniProt Entry Name:	CCR6_HUMAN

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

Human CCR6 ELISA Kit