• Rapidly measure phosphorylated protein in lysates
• Screen numerous different cell lysates without performing a Western Blot analysis
• Minimal hands-on time, convenient, and non-radioactive material

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• Pre-Coated 96-well Strip Microplate
• Wash Buffer
• Biotinylated Anti-Phosphotyrosine Antibody
• Stop Solution
• Assay Diluent(s)
• Positive Control Sample
• Lysis Buffer
• Streptavidin-Conjugated HRP
• TMB One-Step Substrate

• 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
• Shaker
• Microplate reader capable of measuring absorbance at 450 nm
• Absorbent paper
• Log-log graph paper or computer and software for ELISA data analysis

1. Prepare all reagents, samples and positive control as instructed in the manual.
2. Add 100 µL of positive control or sample to each well.
3. Incubate 2.5 hours at room temperature or overnight at 4°C with gentle shaking.
4. Add 100 µL prepared detection antibody to each well.
5. Incubate 1 hour at room temperature with gentle shaking.
6. Add 100 µL prepared HRP-Conjugated solution.
7. Incubate for 1 hour at room temperature with gentle shaking.
8. Add 100 µL TMB One-Step Substrate Reagent to each well.
9. Incubate 30 minutes at room temperature.
10. Add 50 µL 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.