ELISA Protocols

• Sandwich Assay Procedure
• Phosphorylation Assay Procedure
• EIA Assay Procedure
• Cell-based Assay Procedure

Sandwich Assay Procedure

1. Bring all reagents and samples to room temperature (18 - 25 °C) before use. It is recommended that all standards and samples be run at least in duplicate.
2. Add 100 µL of each standard and sample into appropriate wells. Cover wells and incubate for 2.5 hours at room temperature or overnight at 4 °C with gentle shaking.
3. Discard the solution and wash 4 times with 1X Wash Solution. Wash by filling each well with Wash Buffer (300 µL) using a multi-channel Pipette or autowasher. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels.
4. Add 100 µL of 1x prepared Detection Antibody to each well. Cover wells and incubate for 1 hour at room temperature with gentle shaking.
5. Discard the solution. Repeat the wash procedure as in step 3.
6. Add 100 µL of prepared Streptavidin solution to each well. Cover wells and incubate for 45 minutes at room temperature with gentle shaking.
7. Discard the solution. Repeat the wash as in step 3.
8. Add 100 µL of TMB One-Step Substrate Reagent (Item H) to each well. Cover wells and incubate for 30 minutes at room temperature in the dark with gentle shaking.
9. Add 50 µL of Stop Solution (Item I) to each well. Read absorbance at 450 nm immediately.

Phosphorylation Assay Procedure

1. Bring all reagents to room temperature (18 - 25 °C) before use. It is recommended that all samples or Positive Control should be run at least in duplicate.
2. Add 100 µL of each sample or positive control into appropriate wells. Cover well with plate holder and incubate for 2.5 hours at room temperature or overnight at 4 °C with shaking.
3. Discard the solution and wash 4 times with 1x Wash Solution. Wash by filling each well with Wash Buffer (300 µL) using a multi-channel pipette or autowasher. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating. Invert the plate and blot it against clean paper towels.
4. Add 100 µL of prepared 1X biotinylated anti-phosphotyrosine antibody to each well. Incubate for 1 hour at room temperature with shaking.
5. Discard the solution. Repeat the wash as in step 3.
6. Add 100 µL of prepared 1X HRP-Streptavidin solution (see Reagent Preparation step 6) to each well. Incubate for 45 minutes at room temperature with shaking.
7. Discard the solution. Repeat the wash as in step 3.
8. Add 100 µL of TMB One-Step Substrate Reagent (Item H) to each well. Incubate for 30 minutes at room temperature in the dark with shaking.
9. Add 50 µL of Stop Solution (Item I) to each well. Read at 450 nm immediately.