User:Madeleine Y. Bee/Notebook/CHEM-571 2013F/2013/09/24

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Experimental Biological Chemistry: Fall 2013 Main project page
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September 24, 2013

Objective

Pepsin cleavage of hemoglobin according to this procedure.

Description

  1. Combine 5 mL of the hemoglobin solution with the amount of pepsin to make the final concentration 2 nM
    • Do the same only with an amount of pepstatin to make the final concentration 20nM
  2. Incubate these solutions at 37C
  3. After 30 minutes, remove two samples:
    • SDS-PAGE Samples (for TOMORROW)
      1. 10uL of the reaction sample, diluted to 1mL with the Glycine-HCl buffer
      2. 10uL of this diluted sample, mixed with 10uL of the SDS-PAGE running buffer
      3. Store all of your SDS-PAGE samples in the fridge
    • UV-Vis Samples (for TODAY)
      1. 0.75mL of the reaction sample, add 0.75mL of 1M perchloric acid to precipitate the remaining hemoglobin
      2. After 1 hour, centrifuge for 15 minutes to remove solid (uncleaved hemoglobin) from solution
      3. Measure the absorption (specifically note 280nm) to determine the protein concentration in solution
        • Use Glycine-HCl buffer as blank
        • Use Hemoglobin as reference
  4. Repeat Step 3 every 30 minutes for 2 hours.

Data

Image:2013_0924_hemoglobin_abs.PNG

Notes

Gel electrophoresis will be conducted tomorrow.




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