CX3CL1 Kit ELISA

N° du produit ABIN625193

Détail du produit CX3CL1 Kit ELISA

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

Reactivité: Rat

Méthode de détection: Colorimetric

Type de méthode: Sandwich ELISA

Application: ELISA

Fonction: Rat Fractalkine (CX3CL1) ELISA Kit for cell and tissue lysate samples.

Type d'échantillon: Cell Lysate, Tissue Lysate

Analytical Method: Quantitative

Specificité: The antibody pair provided in this kit recognizes rat Fractalkine.

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

Sensibilité: 5 pg/mL

Attributs du produit:

• 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

Ingrédients:

• 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

Matériel non inclus:

• 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
• Cell lysate buffer

Information d'application

Volume d'échantillon: 100 μL

Plaque: Pre-coated

Protocole:

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.

Préparation des réactifs:

1. Bring all reagents and samples to room temperature (18 - 25 °C) before use.
   2. Sample dilution: Tissue lysate and cell lysate sample should be diluted at least 5-fold with 1x Sample Diluent Buffer.
   3. Sample Diluent Buffer (Item D) and Assay Diluent (Item E) should be diluted 5-fold with deionized or distilled water before use.
   4. Preparation of standard: Briefly spin the vial of Item C. Add 400 µL 1x Sample Diluent Buffer (Item D) 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 Sample Diluent Buffer to prepare a 2,000 pg/mL stock standard solution. Pipette 400 µL 1x Sample Diluent Buffer into each tube. Use the stock standard solution to produce a dilution series . Mix each tube thoroughly before the next transfer. 1x Sample Diluent Buffer serves as the zero standard (0 pg/mL). 200 µL 200 µL 200 µL 200myl 200 µL 200 µL 40 µL standard + 960 µL 2000 666.7 222.2 74.07 24.69 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 Diuent 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 Diuent and used in step 4 of Part VI Assay Procedure.
   7. Briefly spin the HRP-Streptavidin concentrate vial (Item G) before use. HRP-Streptavidin concentrate should be diluted 240-fold with 1x Assay Diuent. 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 to prepare a 240-fold diluted HRP- Streptavidin solution (don't store the diluted solution for next day use). Mix well.
   8. Cell lysate buffer should be diluted 2-fold with deionized or distilled water (for cell lysate and tissue lysate).

Procédure de l'essai:

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. We recommend using 50-500 myg/mL of total protein for lysate sample. The amount of sample used depends on the abundance of target protein. More of the sample can be used if signals are too weak. If signals are too strong, the sample can be diluted further.
   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.

Calcul des résultats:

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 Diluent 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 cell lysis buffer. Mean recoveries are as follows: Sample Type Average % Recovery Range ( %) Tissue lysate 92.87 83-103 Cell lysate 93.54 84-104
Linearity: Sample Type Tissue Cell Lysate lysate 1:2 Average % of 89 91 Expected Range ( %) 81-101 82-102 1:4 Average % of 92 96 Expected Range ( %) 84-103 83-103
Reproducibility: Intra-Assay: CV<10 % Inter-Assay: CV<12 %

Précision du teste: Intra-Assay: CV< 10 % Inter-Assay: CV< 12 %

Restrictions: For Research Use only

Stockage

Conseil sur la manipulation: Avoid repeated freeze-thaw cycles.

Stock: -20 °C

Stockage commentaire: 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.

Date de péremption: 6 months

Détail du antigène

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

Autre désignation: Fractalkine / CX3CL1 (CX3CL1 Produits)

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

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

UniProt: Q6IRF7

Pathways: Synaptic Membrane