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If crystals form in the Buffer Concentrates, warm and gently stir them until completely dissolved. Washing Buffer (1x) Pour entire contents (50 mL) of the Washing Buffer (20x) into a clean 1000 mL graduated cylinder. Bring to final volume of 1000 mL with pure or deionized water. Mix gently to avoid foaming. Transfer to a clean wash bottle and store at 2 to 25 °C. Washing Buffer (1x) is stable for 30 days. Assay Buffer (1x) Pour the entire contents (5 mL) of the Assay Buffer (10x) into a clean 100 mL graduated cylinder. Bring to final volume of 50 mL with distilled water. Mix gently to avoid foaming. Store at 2 to 8 °C. Assay Buffer (1x) is stable for 30 days. Detect Antibody Mix well prior to making dilutions. Make a 1:100 dilution of the concentrated Detect Antibody solution with Assay Buffer (1x) in a clean plastic tube as needed. The diluted Detect Antibody should be used within 30 minutes after dilution. Streptavidin-HRP Mix well prior to making dilutions. Make a 1:100 dilution of the concentrated Streptavidin-HRP solution with Assay Buffer (1x) in a clean plastic tube as needed. The diluted Streptavidin-HRP should be used within 30 minutes after dilution. Sample Dilution If your samples have high IL-21 content, dilute serum/plasma samples with Assay Buffer (1x). For cell culture supernates, dilute with cell culture medium. Human IL-21 Standard Reconstitute Human IL-21 Standard by addition of distilled water. Reconstitution volume is stated on the label of the standard vial. Swirl or mix gently to insure complete and homogeneous solubilization (concentration of reconstituted standard = 16000 pg/ mL). Allow the standard to reconstitute for 10 - 30 minutes. Mix well prior to making dilutions. Use polypropylene tubes. Human IL-21 ELISA Kit 7 For serum/plasma samples, mixing concentrated human IL-21 standard (150 μL) with 150 μL of Standard Diluent creates the high standard (8000 pg/ mL). Pipette 150 μL of Standard Diluent into each tube. Use the high standard to produce a 1:1 dilution series . Mix each tube thoroughly before the next transfer. Standard Diluent serves as the zero standard (0 pg/ mL). For cell culture supernates, mixing concentrated human IL-21 standard (150 μL) with 150 μL of cell culture medium creates the high standard (8000 pg/ mL). Pipette 150 μL of cell culture medium into each tube. Use the high standard to produce a 1:1 dilution series. Mix each tube thoroughly before the next transfer. Cell culture medium serves as the zero standard (0 pg/ mL).
Bring all reagents and samples to room temperature before use.1. Prepare all reagents including microplate, samples, standards and working solution as described in the previous sections.2. Remove excess microplate strips and return them to the foil pouch containing the desiccant pack, and reseal for further use.3. Add 300 μL Washing Buffer (1x) per well, and allow it for about 30 seconds before aspiration. Soaking is highly recommended to obtain a good test performance. Empty wells and tap microwell strips on absorbent pad or paper towel to remove excess Washing Buffer (1x). Use the microwell strips immediately after washing. Do not allow wells to dry.4. Add 50 μL of Assay Buffer (1x) to each well.5. Add 50 μL of Standard or sample to each well. Ensure reagent addition is uninterrupted and completed within 15 minutes.6. Add 50 μL of Detect Antibody to each well.7. Seal the plate with an adhesive film. Incubate at room temperature (18 to 25 °C) for 2 hours on a microplate shaker set at 100 rpm.8. Aspirate each well and wash by filling each well with 300 μL Washing Buffer (1x), repeat five times for a total six washes. Complete removal of liquid at each step is essential to the best performance. After the last wash, remove any remaining Washing Buffer (1x) by aspirating or decanting. Invert the plate and tap it against clean paper towels.9. Add 100 μL of Streptavidin-HRP to each well.10. Seal the plate with a fresh adhesive film. Incubate at room temperature (18 to 25 °C) for 45 minutes on a microplate shaker set at 100 rpm.11. Repeat aspiration/wash as in step 8. 12. Add 100 μL of Substrate Solution to each well. Incubate for 10 - 30 minutes at room temperature. Protect from light.13. Add 100 μL of Stop Solution to each well. The color will turn yellow. If the color in the well is green or if the color change does not appear uniform, gently tap the plate to ensure thorough mixing.14. Measure the optical density value within 30 minutes by microplate reader set to 450 nm. If wavelength correction is available, set to 570 nm or 630 nm. If wavelength correction is not available, subtract readings at 570 nm or 630 nm from the readings at 450 nm. This subtraction will correct for optical imperfections in the plate. Reading directly at 450 nm without correction may generate higher concentration than true value.
Average the duplicate optical density readings for each standards and sample, then subtract the average optical density value of the zero standard. Standard Concentration as horizontal axis, optical density (OD) Value as the vertical axis, regressing the data and create a standard curve using computer software. The data may be linearized by plotting the log of the IL-21 concentrations versus the log of the OD and the best fit line can be determined by regression analysis. This procedure will produce an adequate but less precise fit of the data. Note: The finally concentration of top standard is 4000 pg/mL. If instruction in this protocol have been followed samples have been diluted by 1:1 ratio (50 μL sample + 50 μL Assay Buffer), the concentration read from the standard curve must be multiplied by the dilution factor (x2). If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor.