General Block 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.

Reduces background using a mixture of blocking agents, including BSA. General Block contains mammalian protein blocking agents to provide adequate blocking strength for most immunoassays, including monoclonal and polyclonal antibody capture ELISAs and peptide and protein antigen-down ELISAs. This unique blocking buffer contains a heterogeneous mixture of proprietary protein stabilizers and small molecules (including BSA) that block the uncoated regions of the plate. Blocking with ICT’s General Block minimizes non-specific binding interactions during the assay process to reduce background noise and enhance the sensitivity of the assay. General Block provides a microhydrated environment to stabilize the adsorbed protein. This prevents degradation of the coated material and improves retention of protein antigenicity or antibody activity during long-term storage. General Block contains an antimicrobial agent for room temperature blocking of the plate and for long-term storage of the dried plate at 2-8°C. 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. However, allow approximately 10% extra blocking buffer to account for losses during pipetting.