To observe the catalytic activity of pepsin (in cleaving peptide bonds in hemoglobin) in the presence and absence of pepstatin. This data will be compared with data we take in a later lab on pepsin-AuNPs.
The experimental details (pepsin cleavage of hemoglobin) are similar to this reference.
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.
Using C1V1=C2V2, the amount of pepsin used 0.83 uL.
Initial concentration of pepsin is 12 uM. Final concentration is 2 nM. Final volume is 5 mL.
The amount of pepstatin used as well is 8.3 uL as the final concentration is to be 2uM. The initial concentration of pepstatin is 1.2 mM.
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