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|Antigen||Ciliary Neurotrophic Factor (CNTF) ELISA Kits|
|Reactivity||Rat (Rattus) Alternatives|
Kits with alternative reactivity to:
|Method Type||Sandwich ELISA|
|Detection Range||10-2000 pg/mL|
|Minimum Detection Limit||10 pg/mL|
|3 references available|
|Supplier||Log in to see|
Product Details CNTF ELISA KitTarget details Application Details Handling References for CNTF Kit (ABIN625191) Images
|Purpose||Rat CNTF ELISA Kit for cell culture supernatants, plasma, and serum samples.|
|Sample Type||Plasma, Cell Culture Supernatant, Serum|
|Specificity||This ELISA kit shows no cross-reactivity with any of the cytokines tested: rat CINC-2, CINC-3, Fractalkine, 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.|
|Sensitivity||< 10 pg/mL|
|Material not included||
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|Alternative Name||CNTF (CNTF ELISA Kit Abstract)|
|Background||Ciliary neurotrophic factor (CNTF)|
|Research Area||Stem Cells|
Application DetailsProduct Details CNTF ELISA Kit Target details Handling References for CNTF Kit (ABIN625191) Images back to top
|Application Notes||Recommended Dilution for serum and plasma samples2 fold|
|Sample Volume||100 μL|
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: 2 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 and then 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 CNTF 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 200myl 200 µL 200 µL 200 µL 40 µL standard + 960 µL 2000 666.7 222.2 74.07 24.69 8.23 0 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 200-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 10 ml 1x Assay Diluent B to prepare a 200-fold diluted HRP- Streptavidin solution (don't store the diluted solution for next day use). Mix well.
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.
|Calculation of Results||
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 A Rat CNTF concentration (pg/mL) 1 10 100 1000 10000 O D =4 50 (n m ) 0.01 0.1 1 10 Assay Diluent B Rat CNTF 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 CNTF is typically less than 10 pg/mL.
Recovery: Recovery was determined by spiking various levels of Rat CNTF into Rat serum, plasma and cell culture media. Mean recoveries are as follows: Sample Type Average % Recovery Range ( %) Serum 105.6 93-112 Plasma 104.2 92-110 Cell culture media 97.3 89-108
Linearity: Sample Type Serum Plasma Cell Culture Media 1:2 Average % of Expected 95 97 94 Range ( %) 85-104 86-105 82-102 1:4 Average % of Expected 97 101 96 Range ( %) 86-106 90-106 85-104
Reproducibility: Intra-Assay: CV<10 % Inter-Assay: CV<12 %
|Assay Precision||Intra-Assay: CV< 10 % Inter-Assay: CV< 12 %|
|Restrictions||For Research Use only|
HandlingProduct Details CNTF ELISA Kit Target details Application Details References for CNTF Kit (ABIN625191) Images back to top
|Handling Advice||Avoid repeated freeze-thaw cycles.|
|Storage Comment||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.|
|Expiry Date||6 months|
References for CNTF Kit (ABIN625191)Product Details CNTF ELISA Kit Target details Application Details Handling Images back to top
|Product cited in:||
Dinis, Elia, Vidal, Dermigny, Denoeud, Kaplan, Egles, Marin: "3D multi-channel bi-functionalized silk electrospun conduits for peripheral nerve regeneration." in: Journal of the mechanical behavior of biomedical materials, Vol. 41, pp. 43-55, 2015
Bashar, Metcalfe, Yanai, Laver, Hfeli, Gregory-Evans, Moritz, Matsubara, Gregory-Evans: "Influence of Iron Oxide Nanoparticles on Innate and Genetically Modified Secretion Profiles of Mesenchymal Stem Cells." in: IEEE transactions on magnetics, Vol. 49, Issue 1, pp. 389-393, 2014
Yanai, Häfeli, Metcalfe, Soema, Addo, Gregory-Evans, Po, Shan, Moritz, Gregory-Evans: "Focused magnetic stem cell targeting to the retina using superparamagnetic iron oxide nanoparticles." in: Cell transplantation, Vol. 21, Issue 6, pp. 1137-48, 2012
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