Coat and Stabilize Adsorbed Antibodies with CB2
CB2 5x Antigen Coating Buffer maximizes the adsorption of antigens onto polystyrene plates and stabilizes the tertiary structure of the antigen to retain its binding reactivity with the target antibody in the sample.
Enhance Specific Signal, Save Reagents
Stabilizing the coated antigen allows for better analyte detection and enhances the specific signal of the assay. By generating a higher positive signal, a lower concentration of coating antigen may be used, saving valuable reagents.
Extend Shelf Life of Coated Plates
Under proper storage conditions, coated plates may last for years, enabling the manufacture of large batches for inclusion in diagnostic test kits. This also proves useful in non-manufacturing settings, where large batches of plates may be prepared at once to be stored for future experiments.
| Product Datasheet: | |
| Best For: | coating generic proteins ranging from 6kd to > 200kd onto polystyrene plates |
| To Use: | Simply dilute CB2 1:5 and add the coating antigen. Let the solution stir for 15 minutes, pipette onto the plate, and incubate for a few hours. As CB2 is a 5x concentrate, crystalline precipitates may form in the bottle, especially when refrigerated. If this happens, gently warm the buffer until all crystals are dissolved. Do not let it boil. Plate Coating Procedure:
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| pH: | 8.4 at 1x |
| Contains: | No proteins |
| Supplied At: | 5x |
| Storage: | Room Temperature, Ships Overnight (Domestic), 2-Day, 3-Day Shipping Available for orders placed by phone, fax, or email, International Priority Shipping |
| MSDS: | |
| Certificates of Analysis: |
How do I use CB2?
To use CB2, simply dilute it 1:5 and add the coating antigen. Let the solution stir for 15 minutes, pipette onto the plate, and incubate for a few hours. As CB2 is a 5x concentrate, crystalline precipitates may form in the bottle, especially when refrigerated. If this happens, gently warm the buffer until all crystals are dissolved. Do not let it boil.
Which plates should I use?
Be sure to use a 96-well plate specific for ELISA applications, as 96-well tissue culture plates will cause background problems. For antigen-down ELISAs, ICT recommends Immulon® II HB plates, available from ICT (catalog #227), and secondarily Costar® 96-Well EIA/RIA Stripwell™ Plates (ICT catalog #25).
How do I prepare the working solution?
CB2 is supplied as a 5x concentrate to conserve valuable storage space. Simply add 4 parts deionized water to 1 part CB2, mix, and it is ready to use. No pH adjustment is required. During refrigeration, some salts in the buffer may precipitate. Simply warm the bottle in a water bath (do not boil) and mix. The crystals should go into solution.
What are the shelf-lives of the 1x and 5x solutions?
After dilution, the 1x buffer will suppress bacterial growth for up to 1 month at room temperature and 6 months at 2°-8°C. The concentrated 5x buffer will suppress bacterial growth up to 18 months at 2°-8°C.
How much antigen should I use to coat plates?
Generally, plates are coated with 0.1 - 0.8ug/mL of antigen. The concentration depends upon stability of the antigen. ICT recommends performing an initial titration experiment to determine the optimal coating concentration. For example, coat a plate at 4 levels: 1 µg/mL, 2 µg/mL, 4 µg/mL, and 8 µg/mL. When the plate is coated, blocked and ready to use, run a blank and known samples at high, medium, and low concentrations. Look for the wells with the lowest background OD of the blank buffer and highest specific signal. Also consider the differential between the lowest standard and the blank.
How much Coating Buffer do I need?
Generally, plates are coated with 100 µL or 200 µL of antigen coating solution per well. The volume usually depends on the sample volume and the reactivity of the target antibody. For example, if coating 100 µL/well in a 96-well plate, 9.6 mL of 1x coat buffer per plate will be needed. The 25 mL bottle of CB2 provides 125 mL of 1x buffer, which can coat 13 plates. The 100 mL bottle of 5x CB2 will coat 52 plates, and 1L of 5x CB2 will coat 520 plates. ICT also offers each coating buffer in 10L carboys (approx. 5,200 plates).
If coating with 200 µL/well, 19.2 mL of 1x coat buffer per 96-well microtiter plate will be needed. In this way, 6 plates can be coated with the 25 mL trial size, 26 plates can be coated with 100 mL of 5x coat buffer, 130 plates can be coated with 500 mL of 5x CB1, and 260 plates can be coated with the 1L size. ICT also offers each coating buffer in 10L carboys (approx. 2,600 plates).
How do I store the coated plates?
CB2 contains an antimicrobial agent to retard bacterial growth during the plate coating process, allowing the procedure to be performed at room temperature. Once dried, store the coated plates in a foil bag (ICT catalog #6288) to protect the antibody from light, and include a desiccant packet (ICT catalog #6289) to absorb excess moisture, which could degrade the antibody. Refrigerate the plates for long-term storage. When stored at 2°-8°C, the coated antigen should retain its activity for several months or even years. When using the plates, let the plate come to room temperature before opening the bag. This will prevent condensation from collecting in the well and degrading the coated protein.
