- Observe the catalytic activity of pepsin and pepstatin.
- Collect four samples each of pepsin and pepstatin for SDS-PAGE to be run tomorrow.
- Run UV-vis to observe the effects of pepsin and pepstatin.
The following protocol was derived from this reference.
- Combine 5mL hemoglobin with 8.333μL of 1.2E-6M pepsin to make a solution with a final concentration of 2nM.
- Prepare a 5mL sample of hemoglobin with 83.3μL of 1.2mM pepstatin to make a solution with a final concentration of 20μM.
- Incubate these solutions at 37°C
- After 30 minutes, remove the two samples to prepare samples for SDS-PAGE and UV-vis
- SDS-PAGE sample preparation
- Remove 10μL sample and dilute the sample to 1mL with 50mM Glycine-HCl buffer.
- Take 10μL of the diluted sample and add 10μL of SDS-PAGE buffer.
- Store prepared samples in the fridge
- UV-vis sample preparation
- Remove 0.75mL of sample(either pepsin or pepstatin) and add 0.75mL perchloric acid
- Repeat step 4 for two hours.
Figure 1. Absorbance spectra of hemoglobin with pepsin.
Figure 2. Absorbance spectra of hemoglobin with 20μM pepstatin