ELISA Blocking Buffer

1. Coat antibody or antigen onto the ELISA plate using ICT’s Antibody Coating Buffer or Antigen Coating Buffer., 2. Incubate 8–24 hours at room temperature (RT)., 3. Aspirate the coating solution., 4. Wash each Well twice with ICT’s ELISA Wash Buffer., 5. Block the uncoated regions of the ELISA plate by pipetting 300–400 µL of blocking buffer into each Well. Always use an equal or greater volume of blocking buffer than was used for the coating buffer solution., 6. Incubate 8–24 hours at RT. For best blocking, incubate overnight at RT., 7. Aspirate the blocking buffer; do not wash., 8. Run the assay immediately, or dry the plate for long-term storage and seal in a foil storage bag with a desiccant pack. Store dried and packaged plates at 2-8°C.

ELISA Blocking Buffer is designed to avoid false positives associated with animal proteins (e.g., BSA) and eliminate non-specific background noise in antibody-sandwich and antigen-down ELISAs without the use of conventional protein additives. By depositing inert, synthetic blocking molecules onto the plate, ELISA Blocking Buffer reduces non-specific binding of enzyme-labeled conjugates to the microtiter plate, enhancing the sensitivity of the assay. Its synthetic blockers also stabilize coated protein for improved retention of antigen epitope or antibody binding activity during long-term storage. ELISA Blocking Buffer contains an antimicrobial agent for room temperature blocking and long-term storage of dried plates at 2-8°C. ELISA Blocking Buffer works on all types of polystyrene plates except Immulon® II microplates. When blocking with ELISA Blocking Buffer, we recommend Corning® 96-Well EIA/RIA StripWell™ microplates (ICT catalog #25). When preparing plates, the antibody or antigen is typically coated using 50-200 µL of coating solution per Well. After coating, plates are normally washed to remove unbound proteins and then blocked using a larger volume of blocking buffer than was used for coating, such as 300 µL per Well. This ensures that all uncoated regions inside the Well are blocked. A 96-Well plate blocked using this method will require 28.8 mL of blocking solution. Allow approximately 10% extra blocking buffer to account for losses during pipetting.