|Antigen||Interleukin 5 (IL5) Antibodies|
|Reactivity||Mouse (Murine) Alternatives|
|Conjugate||This IL5 antibody is un-conjugated Alternatives|
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Product Details anti-IL5 AntibodyTarget Details IL5 Application Details Handling Images
|Purification||Purified from tissue culture supernatant or ascites by affinity chromatography|
|Sterility||0.2 μm filtered|
|Endotoxin Level||≤0.01 ng/µg|
51-1805KZ: Mouse/Human IL-5 ELISPOT Capture Antibody. Storage Buffer: No azide/low endotoxin: Aqueous buffered solution containing no preservative, 0.2μm filtered. Endotoxin level is ≤0.01 ng/μg of protein.
51-1806KZ: Mouse IL-5 ELISPOT Detection Antibody. Storage Buffer: Aqueous buffered solution containing protein stabilizer and ≤0.09% sodium azide.
Target Details IL5Product Details anti-IL5 Antibody Application Details Handling Images back to top
|Alternative Name||IL-5 (IL5 Antibody Abstract)|
|Background||The enzyme-linked immunospot (ELISPOT) assay is a powerful tool for detecting and enumerating individual cells that secrete a particular protein in vitro. Based on the sandwich ELISA, the ELISPOT assay derives its specificity and sensitivity by employing high affinity capture and detection antibodies and enzyme-amplification. Although originally developed for analyzing specific antibody-secreting cells, the assay has been adapted for measuring the frequencies of cells that produce and secrete other effector molecules, such as cytokines. The sensitivity of the assay lends itself to measurement of even very low frequencies of cytokine producing cells (e.g., 1/300,000). Unique strengths of the assay include high sensitivity, high throughput, high content analysis, minimal volume of biological material required, applicability to frozen/thawed biological samples, and compatibility with other assays. For example, cells analyzed by ELISPOT can be transferred for cloning, proliferation assays, flow cytometry, or other methods of analysis. This product contains sufficient reagents for five 96-well plates, including unlabelled anti-cytokine capture antibody (no azide/low endotoxin format), biotinylated anti-cytokine detection antibody and a Certificate of Analysis that provides lot-specific optimal reagent concentrations|
|Pathways||JAK-STAT Signaling, Positive Regulation of Peptide Hormone Secretion, Production of Molecular Mediator of Immune Response, Feeding Behaviour|
Application DetailsProduct Details anti-IL5 Antibody Target Details IL5 Handling Images back to top
NOTE: Use ELISPOT plates and reagents under aseptic conditions (e.g., using a laminar flow hood or biosafety cabinet) for Steps 1-7.
1. Dilute capture antibody in Coating Buffer (see Certificate of Analysis for antibody dilution information). Add 100 µl of diluted antibody solution to each well of an ELISPOT plate, Millipore plate, Cat. No. S2EM004M99 is recommended.
2. Store plates at 4°C overnight with lid.
3. Discard Coating Antibody. Wash wells 1X with 200 µL/well Blocking Solution.
4. Add 200 µL/well Blocking Solution and incubate for 2 hr at room temperature.
5. Discard Blocking Solution. Prepare mitogen or antigen, diluted in complete tissue culture medium (e.g., RPMI 1640 with FBS, Pen/Strep, and L-glutamine). Add 100 µL/well to ELISPOT plate.
6. Prepare cell suspensions at different densities, (e.g., 1 X 10e5/mL - 2 X 10e6 cells/mL) Add 100 µL/well of each cell suspension to ELISPOT plate wells.
7. Replace ELISPOT plate lid. Incubate ELISPOT plate at 37°C, 5% CO2 and 99% humidity. The duration of the incubation time can be varied (e.g., 2 hr - 24 hr) depending on the nature of the stimulatory cell culture system. Specific activation conditions and incubation times will vary, depending on cell type, kinetics, and cytokine of interest. Please see Certificate of Analysis provided with each ELISPOT Pair for assay conditions of suggested positive controls. Note: After step 7, aseptic conditions are no longer needed.
8. Aspirate cell suspension. Wash wells 2X with deionized (DI) water. Allow wells to soak for 3-5 min at each wash step.
9. Wash wells 3X with 200 µL/well Wash Buffer I. Discard Wash Buffer.
10. Dilute Detection Antibody in Dilution Buffer (see Certificate of Analysis for antibody dilution information). Add 100 µL per well.
11. Replace lid and incubate for 2 hr at room temperature.
12. Discard Detection Antibody solution. Wash wells 3X with 200 µL/well Wash Buffer I.
13. Dilute Streptavidin-Horseradish Peroxidase (HRP) in Dilution Buffer. (BD™ ELISPOT Streptavidin-HRP, Cat. No. 557630 is recommended). Add 100 µL/well diluted Streptavidin-HRP.
14. Replace lid, incubate for 1 hr at room temperature.
15. Discard Streptavidin-HRP solution. Wash wells 4X with 200 µL/well Wash Buffer I.
16. Wash wells 2X with 200 µL/well Wash Buffer II.
17. Add 100 µL of Final Substrate Solution (AEC) to each well. Monitor spot development from 5 - 60 min.
18. Stop substrate reaction by washing wells with DI water.
19. Air-dry plate for 2 hr - overnight at room temperature in the dark, until the plate is completely dry. Store plate in the dark, prior to analysis.
20. Enumerate spots manually using a dissecting microscope or automatically using an ELISPOT Analyzer.
|Restrictions||For Research Use only|
HandlingProduct Details anti-IL5 Antibody Target Details IL5 Application Details Images back to top
|Buffer||Aqueous buffered solution|