ELISA Blocking Buffer - Non-Mammalian-based

1. Prepare samples and controls, 2. Dilute 10X Apoptosis Wash Buffer 1:10 with diH20., 3. Reconstitute FLICA with 50 µL DMSO., 4. Dilute FLICA 1:5 by adding 200 µL PBS., 5. Add diluted FLICA to each sample at 1:30 (e.g., add 10 µL to 290 µL of cultured cells)., 6. Incubate approximately 1 hour., 7. Remove media and wash cells 3 times: add 1X Apoptosis Wash Buffer and spin cells., 8. If desired, label with additional stains, such as Hoechst, Propidium Iodide, 7-AAD, or an antibody., 9. If desired, fix cells., 10. Analyze with a fluorescence microscope, fluorescence plate reader, or flow cytometer. FAM-FLICA excites at 492 nm and emits at 520 nm., , If working with adherent cells, please see the manual for additional protocols.

Reduces background using small, Non-Mammalian-based blocking agents. ELISA Blocking Buffer - Non-Mammalian-based utilizes a Non-Mammalian-based protein extract and small molecular stabilizers to provide a high degree of blocking efficiency. It is designed for antigen-down and antibody sandwich immunoassays with high background problems. ELISA Blocking Buffer - Non-Mammalian-based is a heterogeneous mixture of small molecules capable of blocking the unoccupied regions of the polystyrene plate Wells that are not sterically accessible to larger, traditional mammalian blocking agents. This minimizes non-specific binding interactions and significantly reduces background noise, increasing the sensitivity of the assay. These small blocking molecules also stabilize the adsorbed proteins for improved retention of antigenicity or antibody activity after drying and long-term storage. In addition, the small size of these unique blocking agents results in minimal steric hindrance to key epitope regions of coated proteins, which prevents masking of small coated peptides, enhancing their specific antigenic signal. Since ELISA Blocking Buffer - Non-Mammalian-based utilizes a Non-Mammalian-based protein blocking agent, it is antigenically foreign to most mammalian immune systems. In antigen-down ELISA formats used to detect antigen-specific antibodies, this reduces the possibility of false-positives generated from endogenous antibodies in the sample reacting with plate blocking proteins. ELISA Blocking Buffer - Non-Mammalian-based is particularly useful when working with human, swine, and bovine serum samples, as minimal interaction between the blocking molecules and mammalian serum matrices results in lower backgrounds. ELISA Blocking Buffer - Non-Mammalian-based 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 un-bound 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 sufficiently.