Product Name:	Human C-C motif chemokine 2 (CCL2) ELISA Kit
Product Code:	HUEB0127
Alias:	C-C motif chemokine 2, HC11, Monocyte chemoattractant protein 1, Monocyte chemotactic and activating factor, MCAF, Monocyte chemotactic protein 1, MCP-1, Monocyte secretory protein JE, Small-inducible cytokine A2, CCL2, MCP1, SCYA2
Uniprot:	P13500
Reactivity:	Human
Range:	15.6-1000 pg/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:	CCL2: Chemotactic factor that attracts monocytes and basophils but not neutrophils or eosinophils. Augments monocyte anti-tumor activity. Has been implicated in the pathogenesis of diseases characterized by monocytic infiltrates, like psoriasis, rheumatoid arthritis or atherosclerosis. May be involved in the recruitment of monocytes into the arterial wall during the disease process of atherosclerosis. Monomer or homodimer; in equilibrium. Binds to CCR2 and CCR4. Is tethered on endothelial cells by glycosaminoglycan (GAG) side chains of proteoglycans. Belongs to the intercrine beta (chemokine CC) family.
UniProt Protein Details:	Protein type:Secreted; Chemokine; Secreted, signal peptide; Motility/polarity/chemotaxis Chromosomal Location of Human Ortholog: 17q11.2-q12 Cellular Component: extracellular space; rough endoplasmic reticulum; perinuclear region of cytoplasm; endocytic vesicle; dendrite; extracellular region; synapse; perikaryon; nerve terminal Molecular Function:heparin binding; chemokine activity; CCR2 chemokine receptor binding; receptor binding; protein kinase activity Biological Process: maternal process involved in pregnancy; protein amino acid phosphorylation; response to antibiotic; monocyte chemotaxis; regulation of cell shape; cell surface receptor linked signal transduction; response to vitamin B3; transforming growth factor beta receptor signaling pathway; negative regulation of neuron apoptosis; cellular homeostasis; cell adhesion; neutrophil chemotaxis; organ regeneration; response to amino acid stimulus; JAK-STAT cascade; positive regulation of tumor necrosis factor production; G-protein signaling, coupled to cyclic nucleotide second messenger; organ morphogenesis; unfolded protein response, activation of signaling protein activity; response to ethanol; cellular response to insulin stimulus; response to bacterium; response to heat; response to mechanical stimulus; positive regulation of endothelial cell proliferation; response to activity; response to progesterone stimulus; positive regulation of nitric-oxide synthase biosynthetic process; positive regulation of collagen biosynthetic process; signal transduction; chemotaxis; positive regulation of synaptic transmission; positive regulation of cellular extravasation; protein kinase B signaling cascade; response to wounding; lipopolysaccharide-mediated signaling pathway; response to gamma radiation; angiogenesis; inflammatory response; lymphocyte chemotaxis; aging; unfolded protein response; cytokine and chemokine mediated signaling pathway; MAPKKK cascade; cytoskeleton organization and biogenesis; viral genome replication; macrophage chemotaxis; humoral immune response; leukocyte migration during inflammatory response; cellular calcium ion homeostasis; G-protein coupled receptor protein signaling pathway; negative regulation of angiogenesis; positive regulation of leukocyte mediated cytotoxicity; cellular protein metabolic process; maternal process involved in parturition; response to hypoxia; positive regulation of T cell activation; vascular endothelial growth factor receptor signaling pathway; astrocyte cell migration Disease: Neural Tube Defects; Mycobacterium Tuberculosis, Susceptibility To; Human Immunodeficiency Virus Type 1, Susceptibility To
NCBI Summary:	This gene is one of several cytokine genes clustered on the q-arm of chromosome 17. Chemokines are a superfamily of secreted proteins involved in immunoregulatory and inflammatory processes. The superfamily is divided into four subfamilies based on the arrangement of N-terminal cysteine residues of the mature peptide. This chemokine is a member of the CC subfamily which is characterized by two adjacent cysteine residues. This cytokine displays chemotactic activity for monocytes and basophils but not for neutrophils or eosinophils. It has been implicated in the pathogenesis of diseases characterized by monocytic infiltrates, like psoriasis, rheumatoid arthritis and atherosclerosis. It binds to chemokine receptors CCR2 and CCR4. [provided by RefSeq, Jul 2013]
UniProt Code:	P13500
NCBI GenInfo Identifier:	126842
NCBI Gene ID:	6347
NCBI Accession:	P13500.1
UniProt Secondary Accession:	P13500,Q9UDF3, B2R4V3,
UniProt Related Accession:	P13500
Molecular Weight:	11,025 Da
NCBI Full Name:	C-C motif chemokine 2
NCBI Synonym Full Names:	chemokine (C-C motif) ligand 2
NCBI Official Symbol:	CCL2
NCBI Official Synonym Symbols:	HC11; MCAF; MCP1; MCP-1; SCYA2; GDCF-2; SMC-CF; HSMCR30
NCBI Protein Information:	C-C motif chemokine 2; small-inducible cytokine A2; monocyte secretory protein JE; monocyte chemotactic protein 1; monocyte chemoattractant protein 1; monocyte chemoattractant protein-1; monocyte chemotactic and activating factor; small inducible cytokine subfamily A (Cys-Cys), member 2; small inducible cytokine A2 (monocyte chemotactic protein 1, homologous to mouse Sig-je)
UniProt Protein Name:	C-C motif chemokine 2
UniProt Synonym Protein Names:	HC11; Monocyte chemoattractant protein 1; Monocyte chemotactic and activating factor; MCAF; Monocyte chemotactic protein 1; MCP-1; Monocyte secretory protein JE; Small-inducible cytokine A2
Protein Family:	C-C motif chemokine
UniProt Gene Name:	CCL2
UniProt Entry Name:	CCL2_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.