User:Andrew W Long/Notebook/Protein Modification/2014/06/18: Difference between revisions

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==June 18, 2014==
==June 18, 2014==


"'Overview
'''Overview'''
Solutions that were dialyzed were checked using the spectrophotometer, and it was determined that nearly all protein that was extracted and run through the column was lost during dialysis. Today we are restarting the experiment, and plan to do a few things differently to try to minimize the loss (try manipulating pH, using more dilute myoglobin/buffer solutions, and use different methods/materials for the dialysis).
Solutions that were dialyzed were checked using the spectrophotometer, and it was determined that nearly all protein that was extracted and run through the column was lost during dialysis. Today we are restarting the experiment, and plan to do a few things differently to try to minimize the loss (try manipulating pH, using more dilute myoglobin/buffer solutions, and use different methods/materials for the dialysis).


"'Initial setup:
'''Initial setup:'''
*6 g Tris dissolved into 500 mL H2O and titrated to a pH of 7.2
*6 g Tris dissolved into 500 mL H2O and titrated to a pH of 7.2
*50 mg of myoglobin dissolved into 20 mL of buffer twice (to produce 2 solutions)
*50 mg of myoglobin dissolved into 20 mL of buffer twice (to produce 2 solutions)

Revision as of 09:13, 18 June 2014

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June 18, 2014

Overview Solutions that were dialyzed were checked using the spectrophotometer, and it was determined that nearly all protein that was extracted and run through the column was lost during dialysis. Today we are restarting the experiment, and plan to do a few things differently to try to minimize the loss (try manipulating pH, using more dilute myoglobin/buffer solutions, and use different methods/materials for the dialysis).

Initial setup:

  • 6 g Tris dissolved into 500 mL H2O and titrated to a pH of 7.2
  • 50 mg of myoglobin dissolved into 20 mL of buffer twice (to produce 2 solutions)
  • 20 mL of butanone measured out twice (equal parts butanone for each of the buffer/myoglobin solutions)
  • Ice bucket prepared, buffer/myoglobin buffer solutions and butanone chilled
  • Ring stand and filter funnel apparatus set up
  • Titration calculations done to figure out volume of acid needed to drop each of the buffer/myoglobin solutions to pH 2.5 and 3 respectively
    • This is to see if changing pH will impact extraction process

pH 2.5 Extraction:

  • Once solutions have chilled, HCl is added to one of the solutions to make the pH 2.5, new solution is poured into extraction funnel, and butanone added immediately after. Solution is mixed for 30 seconds.
  • Three distinct layers formed: upper (butanone, deep reddish brown), middle (clear), and lower (aqueous organic)
  • Top layer of butanone was siphoned off using a glass pipette and pipette helper.
    • Saved in a Falcon tube for later spectral analysis
  • Since more than two distinct layers formed, solution must be treated with butanone again, and mixed for 30 more seconds.
  • After a minute, two distinct layers formed: upper (dark red) and lower (clear with a pink tint)
  • One more treatment of butanone is done to ensure total extraction, and mixed for 30 seconds
  • Two layers formed: upper (heme and butanone) lower: (clear but foggy apoprotein layer and Tris buffer)
  • Upper layer siphoned off
  • pH strips used to test the pH of the clear apoprotein fraction, to ensure that it didn't change too dramatically during the extraction process. Rough readings from strips were unclear, so pH probe was used to determine that the final pH of the solution was ___



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