September 24, 2013
Pepsin cleavage of hemoglobin according to this procedure.
- Combine 5 mL of the hemoglobin solution with the amount of pepsin to make the final concentration 2 nM
- Do the same only with an amount of pepstatin to make the final concentration 20nM
- Incubate these solutions at 37C
- After 30 minutes, remove two samples:
- SDS-PAGE Samples (for TOMORROW)
- 10uL of the reaction sample, diluted to 1mL with the Glycine-HCl buffer
- 10uL of this diluted sample, mixed with 10uL of the SDS-PAGE running buffer
- Store all of your SDS-PAGE samples in the fridge
- UV-Vis Samples (for TODAY)
- 0.75mL of the reaction sample, add 0.75mL of 1M perchloric acid to precipitate the remaining hemoglobin
- After 1 hour, centrifuge for 15 minutes to remove solid (uncleaved hemoglobin) from solution
- Measure the absorption (specifically note 280nm) to determine the protein concentration in solution
- Use Glycine-HCl buffer as blank
- Use Hemoglobin as reference
- Repeat Step 3 every 30 minutes for 2 hours.
Gel electrophoresis will be conducted tomorrow.