The two standards provided with each soluble protein Flex Set are provided as a 10X bulk recombinant protein (50,000 pg/mL) when reconstituted in 0.2 mL of assay diluent and should be serially diluted before mixing with the capture beads and the PE-detection reagent for a given assay. Each assay (single bead or multiplex) performed in a given experiment will need to have a standard curve prepared.
1. For multiplex experiments involving 10 or fewer soluble protein Flex Set assays, reconstitute each Flex Set standard vial with 0.2 mL of assay diluent to prepare a 10X bulk standard. Allow the reconstituted standard to equilibrate for at least 15 min before making dilutions. Mix reconstituted protein by pipet only. Do not vortex.
2. Label 12 X 75-mm tubes (BD Falcon, cat. no. 352008) and arrange them in the following order: top standard, 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, and 1:256.
3. Add 100 ¡L of each soluble protein standard to be run in the experiment to the top standard tube.
4. Add assay diluent to the top standard tube to bring the final volume to 1 mL. Example: if five soluble protein Flex Sets are being multiplexed for a given experiment, 100 p,L of each soluble protein Flex Set standard needs to be added to the top standard tube (5 X 100 p.L = 500 p.L total volume) and then add 500 p.L of assay diluent (1 mL assay diluent - 500 p,L [volume of standards added] = 500 p,L assay diluent). Adjust calculations accordingly for multiplexes of 10-20 soluble protein Flex Set assays.
5. Add 500 p,L of assay diluent to each of the remaining tubes.
6. Perform a serial dilution by transferring 500 p,L from the top standard to the 1:2 dilution tube and mix thoroughly. Continue making serial dilutions by transferring 500 p,L from the 1:2 tube to the 1:4 tube and so on to the 1:256 tube and mix thoroughly. Mix by pipet only, do not vortex. Prepare one tube containing assay diluent to serve as negative control.
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