User:Matt Hartings/Notebook/AU Biomaterials Design Lab/2013/09/24: Difference between revisions

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# Combine 5 mL of the hemoglobin solution with the appropriate amount of pepsin (the final concentration should be 2 nM).
# Combine 5 mL of the hemoglobin solution with the appropriate amount of pepsin (the final concentration should be 2 nM).
## Do the same only with an appropriate amount of pepstatin added. (Each group will use a different concentration of pepstatin. We'll want 2 nM, 200 nM, 20uM, 2mM, and 20mM)
## Do the same only with an appropriate amount of pepstatin added. (Each group will use a different concentration of pepstatin. We'll want 2 nM, 200 nM, 20uM, 2mM, and 20mM)
# Incubate these solutions at 37C (in the incubator shaker)
# After 30 minutes, remove 0.75mL and add 0.75mL of 1.7M perchloric acid to precipitate the remaining hemoglobin
## After this sits for 1 hour, centrifuge for 15 minutes (organize centrifuge time with the other groups)
## Measure the absorption spectrum (specifically note 280nm) in order to determine the protein concentration in solution. Use the stock hemoglobin solution as your reference
# Repeat Step 3 every 30 minutes for 2 hours.
# You will need to keep the quenched reactions for running a SDS-PAGE gel tomorrow.


==Data==
==Data==

Revision as of 19:58, 23 September 2013

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Objective

To observe the catalytic activity of pepsin (in cleaving peptide bonds in hemoglobin) in the presence and absence of pepstatin. This data will be compared with data we take in a later lab on pepsin-AuNPs.

Description

The experimental details (pepsin cleavage of hemoglobin) are similar to this reference.

We will allow the pepsin cleavage of hemoglobin to proceed for two hours with aliquots taken and analyzed every 1/2 hour. We will also run parallel reactions with different concentrations of pepstatin (pepsin inhibitor) present. We will save our aliquots for analysis tomorrow using SDS-PAGE.

  1. Combine 5 mL of the hemoglobin solution with the appropriate amount of pepsin (the final concentration should be 2 nM).
    1. Do the same only with an appropriate amount of pepstatin added. (Each group will use a different concentration of pepstatin. We'll want 2 nM, 200 nM, 20uM, 2mM, and 20mM)
  2. Incubate these solutions at 37C (in the incubator shaker)
  3. After 30 minutes, remove 0.75mL and add 0.75mL of 1.7M perchloric acid to precipitate the remaining hemoglobin
    1. After this sits for 1 hour, centrifuge for 15 minutes (organize centrifuge time with the other groups)
    2. Measure the absorption spectrum (specifically note 280nm) in order to determine the protein concentration in solution. Use the stock hemoglobin solution as your reference
  4. Repeat Step 3 every 30 minutes for 2 hours.
  5. You will need to keep the quenched reactions for running a SDS-PAGE gel tomorrow.

Data

  • Add data and results here...

Notes

This area is for any observations or conclusions that you would like to note.


Use categories like tags. Change the "Course" category to the one corresponding to your course. The "Miscellaneous" tag can be used for particular experiments, as instructed by your professor. Please be sure to change or delete this tag as required so that the categories remain well organized.




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