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

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==Date==
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==October 23, 2013==
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Happy Mole Day!
===Objective===
===Objective===
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To measure the activity of pepsin in solution and pepsin-AuNPs in solution.
===Description===
===Description===
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*Make a 37°C water bath to hold at least 3 test tubes (same size as yesterday)
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====Pepsin Digest====
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#Make 3 1mL dilutions for regular pepsin in 0.01N HCl at the following concentrations:
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#* 10μg/mL
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#* 15μg/mL
 +
#* 20μg/mL
 +
#Make 3 1mL dilutions for pepsin-AuNPs in 0.01N HCl at the following concentrations:
 +
#* 10μg/mL
 +
#* 15μg/mL
 +
#* 20μg/mL
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#Make blank for pepsin
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#*2.5mL hemoglobin solution in test tube in 37C bath
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#*5mL perchloric acid
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#*0.5mL pepsin dilution
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#*Remove from bath after 5 minutes
 +
#*Filter
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#*Take UV-vis spectrum
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#Make blank for pepsin-AuNPs
 +
#*2.5mL hemoglobin solution in test tube in 37C bath
 +
#*5mL perchloric acid
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#*0.5mL pepsin-AuNPs
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#*Remove from bath after 5 minutes
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#*Filter
 +
#*Take UV-vis spectrum
 +
#Make test for pepsin
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#*2.5mL hemoglobin solution in test tube in 37C bath
 +
#*0.5mL pepsin dilution
 +
#*Wait 10 minutes
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#*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
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#*Wait 10 minutes
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#*5mL perchloric acid
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#*Remove from bath after 5 minutes
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#*Filter
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#*Take UV-vis spectrum
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#Calculate the number of units/mg of active enzyme in each sample
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#*Units/mg = [A<sub>280</sub>(Test) - A<sub>280</sub>(Blank)]*1000/[10minutes*mg enzyme in reaction mixture]
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====Pepsin-AuNP Analysis====
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(picture)
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<u>Physical Characteristics (by ratio):</u><br.>
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#30-
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#50-
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#70-
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#90-
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#110-
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#130-
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#150-
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#170-
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#190-
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#210-
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<br.>
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*Centrifuge 1mL of each ratio of NPs for 30 minutes
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*Take UV-vis spectrum of each ratio sample
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*Save the sample
===Data===
===Data===

Revision as of 11:44, 23 October 2013

Experimental Biological Chemistry: Fall 2013 Main project page
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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):

  1. 30-
  2. 50-
  3. 70-
  4. 90-
  5. 110-
  6. 130-
  7. 150-
  8. 170-
  9. 190-
  10. 210-


  • 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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