• Microplate reader capable of measuring absorbance at 450 nm
• Shaker
• Pipettes capable of accurately delivering 2 ul to 1 ml volumes
• Pipettes capable of delivering 1 – 25 ml volumes for reagent preparation
• Graduated cylinders: 100 ml and 1 liter
• Distilled or deionized water
• Tubes to prepare sample dilutions

1. Prepare all reagents and samples as instructed.
2. Add 100 µl samples to each well. Incubate 2.5 hours at room temperature or overnight at 4°C.
3. Add 100 µl the prepared 1X Detection antibody solution to each well. Incubate 1 hour.
4. Add 100 µl the prepared 1X HRP-Conjugated Anti-IgG solution to each well. Incubate 1 hour.
5. Add 100 µl TMB One-Step Substrate Reagent to each well. Incubate 30 minutes.
6. Add 50 µl Stop Solution directly to each well (do NOT wash after TMB step).
7. Read at 450 nm immediately.

The kit may be stored at 4°C up to 1 month from the date of shipment. Shelf life can be extended up to 6 months by storing protein (Item F) and antibodies (Items C and D, if included), at -20°C immediately upon delivery. All other kit components should be stored at 4°C.