Rat C-C motif chemokine 2 (Ccl2) ELISA Kit

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SKU:
RTEB0066
Product Type:
ELISA Kit
Size:
96 Assays
Uniprot:
P14844
Range:
15.6-1000 pg/mL
ELISA Type:
Sandwich
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 C-C motif chemokine 2 (Ccl2) ELISA Kit
Product Code:RTEB0066
Alias:C-C motif chemokine 2, Immediate-early serum-responsive protein JE, Monocyte chemoattractant protein 1, Monocyte chemotactic protein 1, MCP-1, Small-inducible cytokine A2, Ccl2, Je, Mcp1, Scya2
Uniprot:P14844
Reactivity:Rat
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: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
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.

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