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ELISA standard curve

Standards and the standard curve

1. Make up a stock solution of 0.08 ug of protein/100 ul of PBS and store at 4 C

To make the stock:

  1. Use protein extracted from fresh hyphae that are nearly 100% immunoreactive. To determine this, run a Bradford and an ELISA assay on the samples and compare concentrations.
  2. If Bradford and ELISA values are nearly the same, make an ELISA curve and test the values by comparing results to a known curve.
  3. Make up 500 ul aliquots of the stock with a concentration of 0.08 ug of protein in 100 ul of PBS or 0.40 ug of protein in 500 ul of PBS.

2. Put 100 ul of the 0.08 ug protein/100 ul of PBS in 2 of the wells and 50 ul PBS in the other ten wells.

3. Transfer 50 ul of the 0.08 ug sample to a neighboring well that has 50 ul PBS.

4. Mix 3-4 times with the micropipette by pulling the sample up and down.

5. Remove 50 ul from these two wells and transfer to 2 adjacent wells. Mix 3-4x. Repeat for these two wells.

6. After the third dilution, remove 50 ul from the two wells that have 100 ul and dispose of it.