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:
- 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.
- If Bradford and ELISA values are nearly the same, make an ELISA curve and test the values by comparing results to a known curve.
- 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.