CX3CL1 ELISA Kit

Produktnummer ABIN1979795

Produktdetails CX3CL1 ELISA Kit

Target: CX3CL1 (Chemokine (C-X3-C Motif) Ligand 1 (CX3CL1))

Reaktivität: Ratte

Nachweismethode: Colorimetric

Methodentyp: Sandwich ELISA

Detektionsbereich: 5-2000 pg/mL

Untere Nachweisgrenze: 5 pg/mL

Applikation: ELISA

Verwendungszweck: Rat Fractalkine (CX3CL1) ELISA Kit for cell culture supernatants, plasma, and serum samples.

Proben: Plasma, Cell Culture Supernatant, Serum

Analytische Methode: Quantitative

Spezifität: This ELISA kit shows no cross-reactivity with any of the cytokines tested: rat CINC-2, CINC-3, CNTF, IL-1 alpha, IL-1 beta, IL-4, IL-6, IL-10, GM-CSF, IFN-gamma, Leptin, Lix, MCP-1, MIP-3 alpha, beta-NGF, TIMP-1, TNF-alpha, VEGF.

Sensitivität: < 5 pg/mL

Produktmerkmale:

• Strip plates and additional reagents allow for use in multiple experiments
• Quantitative protein detection
• Establishes normal range
• The best products for confirmation of antibody array data

Bestandteile:

• Pre-Coated 96-well Strip Microplate
• Wash Buffer
• Stop Solution
• Assay Diluent(s)
• Lyophilized Standard
• Biotinylated Detection Antibody
• Streptavidin-Conjugated HRP
• TMB One-Step Substrate

Benötigtes Material:

• Distilled or deionized water
• Precision pipettes to deliver 2 μL to 1 μL volumes
• Adjustable 1-25 μL pipettes for reagent preparation
• 100 μL and 1 liter graduated cylinders
• Tubes to prepare standard and sample dilutions
• Absorbent paper
• Microplate reader capable of measuring absorbance at 450nm
• Log-log graph paper or computer and software for ELISA data analysis

Anwendungsinformationen

Applikationshinweise: Recommended Dilution for serum and plasma samples3 fold

Probenmenge: 100 μL

Plattentyp: Pre-coated

Protokoll:

1. Prepare all reagents, samples and standards as instructed in the manual.
2. Add 100 μL of standard or sample to each well.
3. Incubate 2.5 h at RT or O/N at 4 °C.
4. Add 100 μL of prepared biotin antibody to each well.
5. Incubate 1 h at RT.
6. Add 100 μL of prepared Streptavidin solution to each well.
7. Incubate 45 min at RT.
8. Add 100 μL of TMB One-Step Substrate Reagent to each well.
9. Incubate 30 min at RT.
10. Add 50 μL of Stop Solution to each well.
11. Read at 450 nm immediately.

Aufbereitung der Reagenzien:

1. Bring all reagents and samples to room temperature (18 - 25 °C) before use.
   2. Sample dilution: If your samples need to be diluted, Assay Diluent A (Item D) should be used for dilution of serum/plasma samples. 1x Assay Diluent B (Item E) should be used for dilution of culture supernatants. Suggested dilution for normal serum/plasma: 3 fold. Please note that levels of the target protein may vary between different specimens. Optimal dilution factors for each sample must be determined by the investigator.
   3. Assay Diluent B should be diluted 5-fold with deionized or distilled water.
   4. Preparation of standard: Briefly spin the vial of Item C. Add 400 µL Assay Diluent A (for serum/plasma samples) or 1x Assay Diluent B (for cell culture medium) into Item C vial to prepare a 50 ng/mL standard. Dissolve the powder thoroughly by a gentle mix. Add 40 µL Fractalkine standard from the vial of Item C, into a tube with 960 µL Assay Diluent A or 1x Assay Diluent B to prepare a 2,000 pg/mL stock standard solution. Pipette 400 µL Assay Diluent A or 1x Assay Diluent B into each tube. Use the stock standard solution to produce a dilution series . Mix each tube thoroughly before the next transfer. Assay Diluent A or 1x Assay Diluent B serves as the zero standard (0 pg/mL). 200 µL 40 µL standard + 960 µL 200myl 200 µL 200 µL 200 µL 200 µL 2000 666.7 222.2 74.07 24.67 8.23 2.74 0 pg/mL pg/mL pg/mL pg/mL pg/mL pg/mL pg/mL pg/mL
   5. If the Wash Concentrate (20x) (Item B) contains visible crystals, warm to room temperature and mix gently until dissolved. Dilute 20 ml of Wash Buffer Concentrate into deionized or distilled water to yield 400 ml of 1x Wash Buffer.
   6. Briefly spin the Detection Antibody vial (Item F) before use. Add 100 µL of 1x Assay Diluent B into the vial to prepare a detection antibody concentrate. Pipette up and down to mix gently (the concentrate can be stored at 4 °C for 5 days). The detection antibody concentrate should be diluted 80-fold with 1x Assay Diluent B and used in step 4 of Part VI Assay Procedure.
   7. Briefly spin the HRP-Streptavidin concentrate vial (Item G) and pipette up and down to mix gently before use. HRP-Streptavidin concentrate should be diluted 240-fold with 1x Assay Diluent B. For example: Briefly spin the vial (Item G) and pipette up and down to mix gently . Add 50 µL of HRP-Streptavidin concentrate into a tube with 12 ml 1x Assay Diluent B to prepare a 240-fold diluted HRP-Streptavidin solution (don't store the diluted solution for next day use). Mix well.

Testdurchführung:

1. Bring all reagents and samples to room temperature (18 - 25 °C) before use. It is recommended that all standards and samples be run at least in duplicate.
   2. Add 100 µL of each standard (see Reagent Preparation step 2) and sample into appropriate wells. Cover well and incubate for 2.5 hours at room temperature or over night at 4 °C with gentle shaking.
   3. Discard the solution and wash 4 times with 1x Wash Solution. Wash by filling each well with Wash Buffer (300 myl) using a multi-channel Pipette or autowasher. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels.
   4. Add 100 µL of 1x prepared biotinylated antibody (Reagent Preparation step 6) to each well. Incubate for 1 hour at room temperature with gentle shaking.
   5. Discard the solution. Repeat the wash as in step
   6. Add 100 µL of prepared Streptavidin solution (see Reagent Preparation step 7) to each well. Incubate for 45 minutes at room temperature with gentle shaking.
   7. Discard the solution. Repeat the wash as in step
   8. Add 100 µL of TMB One-Step Substrate Reagent (Item H) to each well. Incubate for 30 minutes at room temperature in the dark with gentle shaking.
   9. Add 50 µL of Stop Solution (Item I) to each well. Read at 450 nm immediately.

Ergebnisberechnung:

Calculate the mean absorbance for each set of duplicate standards, controls and samples, and subtract the average zero standard optical density. Plot the standard curve on log-log graph paper or using Sigma plot software, with standard concentration on the x-axis and absorbance on the y-axis. Draw the best-fit straight line through the standard points.
Typical Data: These standard curves are for demonstration only. A standard curve must be run with each assay. Assay Buffer A Rat Fractalkine concentration (pg/mL) 1 10 100 1000 10000 O D =4 50 (n m ) 0.01 0.1 1 10 Assay Buffer B Rat Fractalkine concentration (pg/mL) 1 10 100 1000 10000 O D =4 50 (n m ) 0.01 0.1 1 10
Sensitivity: The minimum detectable dose of Fractalkine is typically less than 5 pg/mL.
Recovery: Recovery was determined by spiking various levels of rat Fractalkine into rat serum, plasma and cell culture media. Mean recoveries are as follows: Sample Type Average % Recovery Range ( %) Serum 94.54 83-103 Plasma 93.46 84-105 Cell culture media 93.35 83-104
Linearity: Sample Type Serum Plasma Cell Culture Media 1:2 Average % of Expected 92 94 93 Range ( %) 83-102 84-103 83-104 1:4 Average % of Expected 93 94 95 Range ( %) 84-104 85-104 82-106
Reproducibility: Intra-Assay: CV<10 % Inter-Assay: CV<12 %

Testpräzision: Intra-Assay: CV< 10 % Inter-Assay: CV< 12 %

Beschränkungen: Nur für Forschungszwecke einsetzbar

Handhabung

Handhabung: Avoid repeated freeze-thaw cycles.

Lagerung: -20 °C

Informationen zur Lagerung: The entire kit may be stored at -20°C for up to 1 year from the date of shipment. Avoid repeated freeze-thaw cycles. The kit may be stored at 4°C for up to 6 months. For extended storage, it is recommended to store at -80°C.

Haltbarkeit: 6 months

Referenzen für CX3CL1 ELISA Kit (ABIN1979795)

Koyama, Kotani, Sawamura, Kuribayashi, Konishi, Michinaga: "Different actions of endothelin-1 on chemokine production in rat cultured astrocytes: reduction of CX3CL1/fractalkine and an increase in CCL2/MCP-1 and CXCL1/CINC-1." in: Journal of neuroinflammation, Vol. 10, pp. 51, (2013) (PubMed).

Gada, Scuffham, Griffin, Marwick: "Quality-of-life implications of immediate surgery and watchful waiting in asymptomatic aortic stenosis: a decision-analytic model." in: Circulation. Cardiovascular quality and outcomes, Vol. 4, Issue 5, pp. 541-8, (2012) (PubMed).

Bitar, Al-Mulla: "A defect in Nrf2 signaling constitutes a mechanism for cellular stress hypersensitivity in a genetic rat model of type 2 diabetes." in: American journal of physiology. Endocrinology and metabolism, Vol. 301, Issue 6, pp. E1119-29, (2012) (PubMed).

Uchida, Ito, Nakamura, Igarashi, Oono, Fujimori, Kawabe, Suzuki, Jensen, Takayanagi: "ERK pathway and sheddases play an essential role in ethanol-induced CX3CL1 release in pancreatic stellate cells." in: Laboratory investigation; a journal of technical methods and pathology, Vol. 93, Issue 1, pp. 41-53, (2012) (PubMed).

Bachstetter, Morganti, Jernberg, Schlunk, Mitchell, Brewster, Hudson, Cole, Harrison, Bickford, Gemma: "Fractalkine and CX 3 CR1 regulate hippocampal neurogenesis in adult and aged rats." in: Neurobiology of aging, Vol. 32, Issue 11, pp. 2030-44, (2011) (PubMed).

Clark, Yip, Grist, Gentry, Staniland, Marchand, Dehvari, Wotherspoon, Winter, Ullah, Bevan, Malcangio: "Inhibition of spinal microglial cathepsin S for the reversal of neuropathic pain." in: Proceedings of the National Academy of Sciences of the United States of America, Vol. 104, Issue 25, pp. 10655-60, (2007) (PubMed).

Details zu CX3CL1

Target: CX3CL1 (Chemokine (C-X3-C Motif) Ligand 1 (CX3CL1))

Andere Bezeichnung: Fractalkine / CX3CL1 (CX3CL1 Produkte)

Synonyme: ABCD3 ELISA Kit, CX3CL1 ELISA Kit, DKFZp459K117 ELISA Kit, Cx3c ELISA Kit, Scyd1 ELISA Kit, ABCD-3 ELISA Kit, C3Xkine ELISA Kit, CXC3 ELISA Kit, CXC3C ELISA Kit, NTN ELISA Kit, NTT ELISA Kit, SCYD1 ELISA Kit, fractalkine ELISA Kit, neurotactin ELISA Kit, AB030188 ELISA Kit, AI848747 ELISA Kit, CX3C ELISA Kit, Cxc3 ELISA Kit, D8Bwg0439e ELISA Kit, ATP binding cassette subfamily D member 3 ELISA Kit, C-X3-C motif chemokine ligand 1 ELISA Kit, chemokine (C-X3-C motif) ligand 1 ELISA Kit, ABCD3 ELISA Kit, CX3CL1 ELISA Kit, Cx3cl1 ELISA Kit, Abcd3 ELISA Kit

Hintergrund: Chemokine (C-X3-C motif) ligand 1 (Cx3cl1 protein) (Fractalkine)

UniProt: Q6IRF7

Pathways: Synaptic Membrane