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

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**This is to see if changing pH will impact extraction process
**This is to see if changing pH will impact extraction process
*Once solutions have chilled, correct volumes of HCl are added to solutions to lower pH
*Once solutions have chilled, correct volumes of HCl are added to solutions to lower pH
*HCl added, and butanone added immediately after
*HCl added, new solution is poured into extraction funnel, and butanone added immediately after
*Each of the new solutions are added to their own filter funnels, allowed to rest, and separate layers immediately form
*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.
 
 
*Dark red organic layer is allowed to run through into a beaker, while clear fraction is kept
*Dark red organic layer is allowed to run through into a beaker, while clear fraction is kept
*pH strips used to test the pH of the clear fraction, to ensure that it has remained the same during the extraction process
*pH strips used to test the pH of the clear fraction, to ensure that it has remained the same during the extraction process

Revision as of 08:35, 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).

  • 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
  • Once solutions have chilled, correct volumes of HCl are added to solutions to lower pH
  • HCl added, new solution is poured into extraction funnel, and butanone added immediately after
  • 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.


  • Dark red organic layer is allowed to run through into a beaker, while clear fraction is kept
  • pH strips used to test the pH of the clear fraction, to ensure that it has remained the same during the extraction process



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