User:Madeleine Y. Bee/Notebook/CHEM-571 2013F/2013/10/23: Difference between revisions

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#*Units/mg = [A<sub>280</sub>(Test) - A<sub>280</sub>(Blank)]*1000/[10minutes*mg enzyme in reaction mixture]
#*Units/mg = [A<sub>280</sub>(Test) - A<sub>280</sub>(Blank)]*1000/[10minutes*mg enzyme in reaction mixture]


====Pepsin-AuNP Analysis====
=====Pepsin-AuNP Analysis=====
(picture)
(picture)
<u>Physical Characteristics (by ratio):</u><br.>
<u>Physical Characteristics (by ratio):</u><br.>

Revision as of 08:46, 23 October 2013

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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
  1. Make 3 1mL dilutions for regular pepsin in 0.01N HCl at the following concentrations:
    • 10μg/mL
    • 15μg/mL
    • 20μg/mL
  2. Make 3 1mL dilutions for pepsin-AuNPs in 0.01N HCl at the following concentrations:
    • 10μg/mL
    • 15μg/mL
    • 20μg/mL
  3. 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
  4. 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
  5. 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
  6. 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
  7. 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.>

  1. 30-
  2. 50-
  3. 70-
  4. 90-
  5. 110-
  6. 130-
  7. 150-
  8. 170-
  9. 190-
  10. 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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