Rat MCP-1 / CCL2 ELISA Kit

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SKU:
RTFI00038
Product Type:
ELISA Kit
Size:
96 Assays
Uniprot:
P14844
Sensitivity:
9.375pg/ml
Range:
15.625-1000pg/ml
ELISA Type:
Sandwich ELISA, Double Antibody
Synonyms:
MCP-1, CCL2, GDCF-2, HC11, HSMCR30, MCAF, MCP1, SCYA2, SMC-CF
Reactivity:
Rat
Frequently bought together:

Description

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Product Name:Rat MCP-1 (Monocyte Chemotactic Protein 1) ELISA Kit
Product Code:RTFI00038
Size:96 Assays
Target:Rat MCP-1
Alias:MCP-1, CCL2, GDCF-2, HC11, HSMCR30, MCAF, MCP1, SCYA2, SMC-CF
Reactivity:Rat
Detection Method:Sandwich ELISA, Double Antibody
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 Rat MCP-1 and the recovery rates were calculated by comparing the measured value to the expected amount of Rat MCP-1 in samples.
MatrixRecovery range(%)Average(%)
serum(n=5)85-9691
EDTA plasma(n=5)85-10492
UFH plasma(n=5)87-10296
Linearity:The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Rat MCP-1 and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.
Sample1:21:41:8
serum(n=5)86-102%86-104%87-105%
EDTA plasma(n=5)84-98%87-97%89-99%
UFH plasma(n=5)82-93%89-97%84-98%
Intra-Assay:CV <8%
Inter-Assay:CV <10%
Uniprot:P14844
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, signal peptide; Secreted; Motility/polarity/chemotaxis; Chemokine

Cellular Component: extracellular space; rough endoplasmic reticulum; cell soma; perinuclear region of cytoplasm; endocytic vesicle; cytoplasm; dendrite; extracellular region; synapse; perikaryon; nerve terminal

Molecular Function:heparin binding; G-protein-coupled receptor binding; chemokine activity; CCR2 chemokine receptor binding

Biological Process: positive regulation of cell adhesion; maternal process involved in pregnancy; positive regulation of leukocyte migration; response to glucocorticoid stimulus; response to lipopolysaccharide; response to antibiotic; monocyte chemotaxis; regulation of cell shape; transforming growth factor beta receptor signaling pathway; response to vitamin B3; negative regulation of neuron apoptosis; response to drug; neutrophil chemotaxis; organ regeneration; response to amino acid stimulus; positive regulation of tumor necrosis factor production; 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; chemokinesis; positive regulation of synaptic transmission; positive regulation of cellular extravasation; positive regulation of cell-cell adhesion; protein kinase B signaling cascade; response to wounding; lipopolysaccharide-mediated signaling pathway; response to gamma radiation; inflammatory response; lymphocyte chemotaxis; aging; cytokine and chemokine mediated signaling pathway; MAPKKK cascade; cytoskeleton organization and biogenesis; glial cell migration; macrophage chemotaxis; leukocyte migration during inflammatory response; cellular calcium ion homeostasis; positive regulation of leukocyte mediated cytotoxicity; negative regulation of angiogenesis; maternal process involved in parturition; response to hypoxia; positive regulation of T cell activation; vascular endothelial growth factor receptor signaling pathway; astrocyte cell migration

NCBI Summary:a monocyte chemoattractant protein [RGD, Feb 2006]
UniProt Code:P14844
NCBI GenInfo Identifier:126846
NCBI Gene ID:24770
NCBI Accession:P14844.1
UniProt Secondary Accession:P14844,Q549R5,
UniProt Related Accession:P14844
Molecular Weight:16,460 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:MCP-1; Scya2; Sigje  
NCBI Protein Information:C-C motif chemokine 2; small inducible gene JE; small inducible cytokine A2; small-inducible cytokine A2; monocyte chemotactic protein 1; monocyte chemoattractant protein 1; monocyte chemoattractant protein-1; immediate-early serum-responsive JE protein; immediate-early serum-responsive protein JE
UniProt Protein Name:C-C motif chemokine 2
UniProt Synonym Protein Names:Immediate-early serum-responsive protein JE; Monocyte chemoattractant protein 1; Monocyte chemotactic protein 1; MCP-1; Small-inducible cytokine A2
Protein Family:C-C motif chemokine
UniProt Gene Name:Ccl2  
UniProt Entry Name:CCL2_RAT
StepProcedure
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 clotovernight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Removeserum 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 anti-coagulant. 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 & homogenizein 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.

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