Substance P Assay Ki (1 Kit)

Assay Procedure

1.   Prepare all reagents, standard dilutions, and samples as directed in the product insert.
2.   Remove excess microplate strips from the plate frame, return them to the foil pouch containing the desiccant pack, and reseal.

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Calibrator Diluent

3.   Add 100 µL of Calibrator Diluent to the non-specific binding (NSB) wells.
4.   Add 50 µL of Calibrator Diluent to the zero standard (B₀) wells.

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50 µL Standard, Control, or Sample

5.   Add 50 µL of Standard, Control, or sample to the remaining wells.

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50 µL Primary Antibody Solution

6.   Add 50 µL of Primary Antibody Solution to each well (except the NSB wells).

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50 µL Conjugate

7.   Add 50 µL of Conjugate to each well. Cover with a plate sealer, and incubate at room temperature for 3 hours on a horizontal orbital microplate shaker.
8.   Aspirate each well and wash, repeating the process 3 times for a total of 4 washes.

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200 µL Substrate Solution

9.   Add 200 µL Substrate Solution to each well. Incubate at room temperature for 30 minutes on the benchtop. PROTECT FROM LIGHT.

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50 µL Stop Solution

10. Add 50 µL of Stop Solution to each well. Read at 450 nm within 30 minutes. Set wavelength correction to 540 nm or 570 nm.