• Simultaneously measure phosphorylated protein and pan protein in one experiment (for normalization purpose)
• Screen numerous different cell lysates without performing a Western Blot analysis
• Minimal hands-on time, convenient, and non-radioactive material

• 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.

Upon receipt, the kit should be stored at -20 °C. Please use within 6 months from the date of shipment. After initial use, Wash Buffer Concentrate (Item B), Assay Diluent (Item E), TMB One-Step Substrate Reagent (Item H), HRP-Streptavidin (Item G), Stop Solution (Item I) and Cell Lysate Buffer (Item J) should be stored at 4 °C to avoid repeated freeze-thaw cycles. Return unused wells to the pouch containing desiccant pack, reseal along entire edge and store at -20 °C. Reconstituted Positive Control (Item K) should be stored at -70 °C.