User:Moira M. Esson/Notebook/CHEM-571/2013/09/24: Difference between revisions

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##*Remove 0.75mL of sample(either pepsin or pepstatin) and add 0.75mL perchloric acid
##*Remove 0.75mL of sample(either pepsin or pepstatin) and add 0.75mL perchloric acid
#Repeat step 4 for two hours.
#Repeat step 4 for two hours.
<br>
==UV-vis==
Figure 1. Absorbance spectra of hemoglobin with pepsin.
<br>
[[Image:Pepsin09242013zem.png]]
<br>
Figure 2. Absorbance spectra of hemoglobin with 20μM pepstatin
<br>
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Revision as of 12:50, 24 September 2013

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Objectives

  1. Observe the catalytic activity of pepsin and pepstatin.
  2. Collect four samples each of pepsin and pepstatin for SDS-PAGE to be run tomorrow.
  3. Run UV-vis to observe the effects of pepsin and pepstatin.


General Protocol

The following protocol was derived from this reference.

  1. Combine 5mL hemoglobin with 8.333μL of 1.2E-6M pepsin to make a solution with a final concentration of 2nM.
  2. Prepare a 5mL sample of hemoglobin with 83.3μL of 1.2mM pepstatin to make a solution with a final concentration of 20μM.
  3. Incubate these solutions at 37°C
  4. After 30 minutes, remove the two samples to prepare samples for SDS-PAGE and UV-vis
    1. 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
    2. UV-vis sample preparation
      • Remove 0.75mL of sample(either pepsin or pepstatin) and add 0.75mL perchloric acid
  5. Repeat step 4 for two hours.


UV-vis

Figure 1. Absorbance spectra of hemoglobin with pepsin.

Figure 2. Absorbance spectra of hemoglobin with 20μM pepstatin
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