October 23, 2013
Happy Mole Day!
Objective
To measure the activity of pepsin in solution and pepsin-AuNPs in solution.
Description
- Make a 37°C water bath to hold at least 3 test tubes (same size as yesterday)
Pepsin Digest
- Make 3 1mL dilutions for regular pepsin in 0.01N HCl at the following concentrations:
- Make 3 1mL dilutions for pepsin-AuNPs in 0.01N HCl at the following concentrations:
- Make blank for pepsin
- 2.5mL hemoglobin solution in test tube in 37C bath
- 5mL perchloric acid
- 0.5mL pepsin dilution
- Remove from bath after 5 minutes
- Filter
- Take UV-vis spectrum
- Make blank for pepsin-AuNPs
- 2.5mL hemoglobin solution in test tube in 37C bath
- 5mL perchloric acid
- 0.5mL pepsin-AuNPs
- Remove from bath after 5 minutes
- Filter
- Take UV-vis spectrum
- Make test for pepsin
- 2.5mL hemoglobin solution in test tube in 37C bath
- 0.5mL pepsin dilution
- Wait 10 minutes
- 5mL perchloric acid
- Remove from bath after 5 minutes
- Filter
- Take UV-vis spectrum
- Make test for pepsin-AuNPs
- 2.5mL hemoglobin solution in test tube in 37C bath
- 0.5mL pepsin-AuNPs
- Wait 10 minutes
- 5mL perchloric acid
- Remove from bath after 5 minutes
- Filter
- Take UV-vis spectrum
- Calculate the number of units/mg of active enzyme in each sample
- Units/mg = [A280(Test) - A280(Blank)]*1000/[10minutes*mg enzyme in reaction mixture]
Pepsin-AuNP Analysis
(picture)
Physical Characteristics (by ratio):<br.>
- 30-
- 50-
- 70-
- 90-
- 110-
- 130-
- 150-
- 170-
- 190-
- 210-
<br.>
- Centrifuge 1mL of each ratio of NPs for 30 minutes
- Take UV-vis spectrum of each ratio sample
- Save the sample
Data
Notes
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