• Pre-Coated 96-well Strip Microplate
• Wash Buffer
• Anti-Phospho Antibody
• Anti-Pan Antibody (*not included in phospho-only version)
• HRP-Conjugated Secondary Antibody
• Assay Diluent
• TMB One-Step Substrate
• Stop Solution
• Lysis Buffer
• Positive Control Sample

• Distilled or deionized water
• 100 ml and 1 liter graduated cylinders
• Tubes to prepare sample dilutions
• Protease and Phosphatase inhibitors
• Precision pipettes to deliver 2 µl to 1 ml volumes
• Adjustable 1-25 ml pipettes for reagent preparation
• Benchtop rocker or shaker
• Microplate reader capable of measuring absorbance at 450 nm

1. Prepare all reagents and samples as instructed in the manual.
2. Add 100 µl of sample or positive control to each well.
3. Incubate 2.5 h at RT or O/N at 4 °C.
4. Add 100 µl of prepared primary antibody to each well.
5. Incubate 1 h at RT.
6. Add 100 µl of prepared 1X HRP-Streptavidin to each well.
7. Incubate 1 h at RT.
8. Add 100 µl of TMB One-Step Substrate Reagent to each well.
9. Incubate 30 min at RT.
10. Add 50 µl of Stop Solution to each well.
11. Read at 450 nm immediately.

HeLa cells were treated with Anisomycin. Solubilize cells at 4 x 10⁷ cells/ml in Cell Lysate Buffer. Serial dilutions of lysates were analyzed in this ELISA. Please see step 3 of Part VI Reagent Preparation for detail.

HeLa cells were treated with Anisomycin. Cell lysates were analyzed using this phosphoELISA and Western Blot.