The following protocol was taken from [User:Matt Hartings/Notebook/AU Biomaterials Design Lab/2013/09/24 Matt Hartings]
We will allow the pepsin cleavage of hemoglobin to proceed for two hours with aliquots taken and analyzed every 1/2 hour. We will also run parallel reactions with different concentrations of pepstatin (pepsin inhibitor) present. We will save our aliquots for analysis tomorrow using SDS-PAGE.
- Combine 5 mL of the hemoglobin solution with the appropriate amount of pepsin (the final concentration should be 2 nM).
- Do the same only with an appropriate amount of pepstatin added. (Each group will use a different concentration of pepstatin. We'll want 2 nM, 20 nM, 0.2uM, 2uM, and 20uM)
- Incubate these solutions at 37C (in the incubator shaker)
- After 30 minutes, remove two samples, as instructed below, for analysis today and tomorrow.
- Prepare a sample for SDS-PAGE tomorrow
- Remove 10uL of the reaction sample and dilute to 1mL with the Glycine-HCl buffer
- Take 10uL of this diluted sample and mix with 10uL of the SDS-PAGE running buffer
- Store all of your SDS-PAGE samples in the fridge overnight.
- Prepare a sample for UV-Vis analysis today
- Remove 0.75mL of the reaction sample and add 0.75mL of 1.7M perchloric acid to precipitate the remaining hemoglobin
- After this sample sits for 1 hour, centrifuge for 15 minutes (organize centrifuge time with the other groups) in order to remove solid (uncleaved hemoglobin) from solution
- Measure the absorption spectrum (specifically note 280nm) in order to determine the protein concentration in solution. Use the stock hemoglobin solution as your reference
- Repeat Step 3 every 30 minutes for 2 hours.