Product Name:	Mouse MCP-1 / CCL2 ELISA Kit
Product Code:	MOFI00072
Size:	96 Assays
Alias:	MCP-1, CCL2, GDCF-2, HC11, HSMCR30, MCAF, MCP1, SCYA2, SMC-CF
Detection Method:	Sandwich ELISA
Application:	This immunoassay kit allows for the in vitro quantitative determination of Mouse MCP-1 concentrations in serum plasma and other biological fluids.
Sensitivity:	9.375pg/ml
Range:	15.625-1000pg/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:

Matrices listed below were spiked with certain level of Mouse MCP-1 and the recovery rates were calculated by comparing the measured value to the expected amount of Mouse MCP-1 in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	88-103	94
EDTA plasma(n=5)	85-104	96
UFH plasma(n=5)	86-104	93

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Mouse MCP-1 and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.

Sample	1:2	1:4	1:8
serum(n=5)	85-100%	85-104%	88-104%
EDTA plasma(n=5)	85-98%	86-101%	90-98%
UFH plasma(n=5)	80-97%	86-97%	85-98%

Intra Assay:

CV <8%

Inter Assay:

CV <10%

Component	Quantity	Storage
ELISA Microplate (Dismountable)	8×12 strips	4°C for 6 months
Lyophilized Standard	2	4°C/-20°C
Sample/Standard Dilution Buffer	20ml	4°C
Biotin-labeled Antibody(Concentrated)	120ul	4°C (Protect from light)
Antibody Dilution Buffer	10ml	4°C
HRP-Streptavidin Conjugate(SABC)	120ul	4°C (Protect from light)
SABC Dilution Buffer	10ml	4°C
TMB Substrate	10ml	4°C (Protect from light)
Stop Solution	10ml	4°C
Wash Buffer(25X)	30ml	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

Uniprot

P10148

UniProt Protein Function:	CCL13: Chemotactic factor that attracts monocytes, lymphocytes, basophils and eosinophils, but not neutrophils. Signals through CCR2B and CCR3 receptors. Plays a role in the accumulation of leukocytes at both sides of allergic and non-allergic inflammation. May be involved in the recruitment of monocytes into the arterial wall during the disease process of atherosclerosis. May play a role in the monocyte attraction in tissues chronically exposed to exogenous pathogens. By IL1/interleukin-1 and TNF. Widely expressed. Found in small intestine, thymus, colon, lung, trachea, stomach and lymph node. Low levels seen in the pulmonary artery smooth muscle cells. Belongs to the intercrine beta (chemokine CC) family.
UniProt Protein Details:	Protein type:Secreted; Secreted, signal peptide; Motility/polarity/chemotaxis; Chemokine Cellular Component: extracellular space; rough endoplasmic reticulum; cell soma; perinuclear region of cytoplasm; endocytic vesicle; dendrite; cytoplasm; extracellular region; perikaryon; synapse; nerve terminal Molecular Function:heparin binding; protein binding; G-protein-coupled receptor binding; chemokine activity; CCR2 chemokine receptor binding; cytokine activity Biological Process: positive regulation of cell adhesion; positive regulation of leukocyte migration; positive regulation of collagen biosynthetic process; chemotaxis; positive regulation of synaptic transmission; positive regulation of cellular extravasation; regulation of cell shape; transforming growth factor beta receptor signaling pathway; positive regulation of cell-cell adhesion; response to wounding; angiogenesis; inflammatory response; lymphocyte chemotaxis; neutrophil chemotaxis; cytokine and chemokine mediated signaling pathway; cytoskeleton organization and biogenesis; positive regulation of tumor necrosis factor production; glial cell migration; macrophage chemotaxis; leukocyte migration during inflammatory response; cellular calcium ion homeostasis; negative regulation of angiogenesis; positive regulation of leukocyte mediated cytotoxicity; response to heat; eosinophil chemotaxis; immune response; positive regulation of endothelial cell proliferation; positive regulation of T cell activation; vascular endothelial growth factor receptor signaling pathway; positive regulation of nitric-oxide synthase biosynthetic process
NCBI Summary:	This gene is one of several cytokine genes clustered on chromosome 11. 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 memory T cells but not for neutrophils. The human ortholog has been implicated in the pathogenesis of diseases characterized by monocytic infiltrates, such as psoriasis, rheumatoid arthritis, and atherosclerosis. [provided by RefSeq, Sep 2015]
UniProt Code:	P10148
NCBI GenInfo Identifier:	126844
NCBI Gene ID:	20296
NCBI Accession:	P10148.1
UniProt Secondary Accession:	P10148,Q9QYD7,
UniProt Related Accession:	P10148
Molecular Weight:	16,326 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:	JE; HC11; MCAF; MCP1; MCP-1; Scya2; Sigje; SMC-CF; AI323594
NCBI Protein Information:	C-C motif chemokine 2; small inducible cytokine A2; small-inducible cytokine A2; monocyte chemotactic protein 1; monocyte chemoattractant protein 1; monocyte chemoattractant protein-1; platelet-derived growth factor-inducible protein JE
UniProt Protein Name:	C-C motif chemokine 2
UniProt Synonym Protein Names:	Monocyte chemoattractant protein 1; Monocyte chemotactic protein 1; MCP-1; Platelet-derived growth factor-inducible protein JE; Small-inducible cytokine A2
Protein Family:	C-C motif chemokine
UniProt Gene Name:	Ccl2
UniProt Entry Name:	CCL2_MOUSE

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

Step	Procedure
1.	Set standard, test sample and control (zero) wells on the pre-coated plate respectively, and then, record their positions. It is recommended to measure each standard and sample in duplicate. Wash plate 2 times before adding standard, sample and control (zero) wells!
2.	Aliquot 0.1ml standard solutions into the standard wells.
3.	Add 0.1 ml of Sample / Standard dilution buffer into the control (zero) well.
4.	Add 0.1 ml of properly diluted sample (Human serum, plasma, tissue homogenates and other biological fluids.) into test sample wells.
5.	Seal the plate with a cover and incubate at 37 °C for 90 min.
6.	Remove the cover and discard the plate content, clap the plate on the absorbent filter papers or other absorbent material. Do NOT let the wells completely dry at any time. Wash plate X2.
7.	Add 0.1 ml of Biotin- detection antibody working solution into the above wells (standard, test sample & zero wells). Add the solution at the bottom of each well without touching the side wall.
8.	Seal the plate with a cover and incubate at 37°C for 60 min.
9.	Remove the cover, and wash plate 3 times with Wash buffer. Let wash buffer rest in wells for 1 min between each wash.
10.	Add 0.1 ml of SABC working solution into each well, cover the plate and incubate at 37°C for 30 min.
11.	Remove the cover and wash plate 5 times with Wash buffer, and each time let the wash buffer stay in the wells for 1-2 min.
12.	Add 90 µL of TMB substrate into each well, cover the plate and incubate at 37°C in dark within 10-20 min. (Note: This incubation time is for reference use only, the optimal time should be determined by end user.) And the shades of blue can be seen in the first 3-4 wells (with most concentrated standard solutions), the other wells show no obvious color.
13.	Add 50 µL of Stop solution into each well and mix thoroughly. The color changes into yellow immediately.
14.	Read the O.D. absorbance at 450 nm in a microplate reader immediately after adding the stop solution.

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.

Antibodies	ELISA
PPBP Antibody, Biotin conjugated	Mouse MCP-1 (Monocyte Chemotactic Protein 1) CLIA Kit
Ccl2 Antibody	Mouse C-C motif chemokine 2 (Ccl2) ELISA Kit
Ccl2 Antibody, HRP conjugated
Ccl2 Antibody, FITC conjugated