Rat CCR2(C-C chemokine receptor type 2) ELISA Kit

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SKU:
RTFI01291
€499

Description

ELISA Kit Technical ManualELISA Kit Technical ManualMSDS

Product name

Rat CCR2 (C-C chemokine receptor type 2) ELISA Kit

Catalogue No.

RTFI01291

Alias

CCR2 ELISA Kit

Detection method

Sandwich ELISA, Double Antibody

Size

96T

Range

15.6-1000pg/ml

Sensitivity

< 3.9pg/ml

Storage

4'C for 6 months

Recovery

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

Matrix Recovery range(%) Average(%)
serum(n=5) 89-103 96
EDTA plasma(n=5) 88-102 95
heparin plasma(n=5) 88-103 96
Linearity

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

Sample 1:2 1:4 1:8 1:16
serum(n=5) 86-91% 85-103% 90-105% 86-103%
EDTA plasma(n=5) 92-101% 89-101% 82-99% 82-97%
heparin plasma(n=5) 81-99% 80-97% 91-98% 82-97%
CV(%)

Intra-Assay: CV<8%
Inter-Assay: CV<10%

Note

For Research Use Only

Rat CCR2(C-C chemokine receptor type 2) ELISA Kit Protocol

The below protocol is a sample protocol for Rat CCR2(C-C chemokine receptor type 2) ELISA Kit using a biotinylated detection antibody and streptavidin-HRP. Sandwich ELISAs allow for the detection and quantification of an analyte in a sample by using known analyte concentrations as standards and plotting absorbance of known concentrations vs known standard concentrations. This allows the researcher to calculate the amount of Rat CCR2(C-C chemokine receptor type 2) present in their sample.

Before adding to wells, equilibrate the SABC working solution and TMB substrate for at least 30 min at 37 ‚°C. When diluting samples and reagents, they must be mixed completely and evenly. It is recommended to plot a standard curve for each test.

Sandwich Protocol

Sandwich ELISA Protocol

Kit Protocol:

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.

Rat CCR2(C-C chemokine receptor type 2) ELISA Kit components

96 Assays

Storage

ELISA Microplate(Dismountable)8×12 strips4°C for 6 months
Lyophilized Standard24°C/-20°C
Sample/Standard Dilution Buffer20ml4°C
Biotin-labeled Antibody(Concentrated)120ul4°C (Protect from light)
Antibody Dilution Buffer10ml4°C
HRP-Streptavidin Conjugate(SABC)120ul4°C (Protect from light)
SABC Dilution Buffer10ml4°C
TMB Substrate10ml4°C (Protect from light)
Stop Solution10ml4°C
Wash Buffer(25X)30ml4°C
Plate Sealer5 -

Other materials and equipment required:

The ELISA Genie Rat CCR2(C-C chemokine receptor type 2) ELISA Kit will require other equipment and materials to carry out the assay. Please see list below for further details.
  • 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

Sample Preparation

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 TypeProtocol

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.

Rat CCR2(C-C chemokine receptor type 2) ELISA Kit Protein Information

UniProt Protein Function:CCR2: a seven transmembrane protein closely related to CCR1. Receptor for the MCP-1, MCP-3 and MCP-4 chemokines. Expressed at high levels in primary neutrophils and primary monocytes, and is further upregulated on neutrophil activation and during monocyte to macrophage differentiation. Transduces a signal by increasing the intracellular calcium ions level. Alternative co receptor with CD4 for HIV-1 infection. Two splice-variant isoforms have been described.
UniProt Protein Details:

Protein type:GPCR, family 1; Membrane protein, integral; Membrane protein, multi-pass; Receptor, GPCR

Chromosomal Location of Human Ortholog: 8q32

Cellular Component: cell soma; cytoplasm; cytosol; dendrite; external side of plasma membrane; perikaryon; perinuclear region of cytoplasm; plasma membrane

Molecular Function:C-C chemokine binding; C-C chemokine receptor activity; CCR2 chemokine receptor binding; chemokine binding; cytokine binding; protein homodimerization activity

Biological Process: blood vessel remodeling; cellular calcium ion homeostasis; cellular defense response; cellular homeostasis; cytokine and chemokine mediated signaling pathway; G-protein coupled receptor protein signaling pathway; hemopoiesis; homeostasis of number of cells within a tissue; humoral immune response; immune response; inflammatory response; inflammatory response to antigenic stimulus; monocyte chemotaxis; negative regulation of angiogenesis; negative regulation of eosinophil degranulation; negative regulation of T-helper 2 type immune response; positive regulation of alpha-beta T cell proliferation; positive regulation of inflammatory response; positive regulation of interferon-gamma production; positive regulation of interleukin-2 production; positive regulation of MAPKKK cascade; positive regulation of T cell activation; positive regulation of T-helper 1 type immune response; positive regulation of tumor necrosis factor biosynthetic process; positive regulation of tumor necrosis factor production; regulation of cell migration; response to hyperoxia; response to hypoxia; sensory perception of pain

NCBI Summary:mediates leukocyte migration and activation [RGD, Feb 2006]
UniProt Code:O55193
NCBI GenInfo Identifier:10719941
NCBI Gene ID:60463
NCBI Accession:O55193.1
UniProt Related Accession:O55193
Molecular Weight:42,763 Da
NCBI Full Name:C-C chemokine receptor type 2
NCBI Synonym Full Names:C-C motif chemokine receptor 2
NCBI Official Symbol:Ccr2  
NCBI Protein Information:C-C chemokine receptor type 2
UniProt Protein Name:C-C chemokine receptor type 2
UniProt Synonym Protein Names:CD_antigen: CD192
Protein Family:C-C chemokine receptor
UniProt Gene Name:Ccr2  
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Additional Information

Product type:
ELISA
Reactivity:
Rat
ELISA Type:
Sandwich
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