User:Tamra L. Fisher/Notebook/Experimental Biological Chem/2011/11/15

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Objective

Play with stuff, see what happens.

Description

Resuspending Protein Fibers

  • Transfer previously made purple protein fibers with tweezers and place into approximately 1mL of 6M NaOH.
  • Remove all solution from a microcentrifuge tube with protein fibers, and resuspend the protein fibers in approximately 1mL of 6M NaOH.
  • Remove all solution from a 10mL test tube with protein fibers, and resuspend the protein fibers in approximately 4mL of ethanol.
  • Centrifuge a previously made purple solution at 13200rpm for 25 minutes to form a pellet. Because the pellet kept falling apart 1.5M Tris at pH 7.5 was added

Refolding Protein

  1. 25μL of 4.17mM HAuCl4 and 50μL of 30μM BSA were combined with 950μL of distilled water in a 1.5 mL microcentrifuge tube.
  2. This tube was placed on a heat block at 80°C for approximately 1.5hrs.
  3. A spectrum of the solution was taken.
  4. 0.97mL of 1.5M Tris buffer, pH 7.5 was added to the solution. A spectrum was taken.
  5. An additional 0.97mL of 1.5M Tris buffer, pH 7.5 was added to the solution. A spectrum was taken.

Making More Protein Fibers

  1. 25μL of 4.17mM HAuCl4 and 25μL of 30μM BSA were combined with 950μL of distilled water in a 1.5 mL microcentrifuge tube.
  2. This tube was placed on a heat block at 80°C for approximately 1.5hrs.

Data

Resuspending Protein Fibers

  • The two methods of suspending purple protein fibers in 6M NaOH did not show any immediate changes to the fibers. After letting the fibers sit in the base overnight the fibers dissolved. The solutions with dissolved protein fibers had a light pinkish-purplish color.
  • The fibers that were resuspended in ethanol did not appear to change, even after remaining in the liquid overnight.
  • Nothing happened to the pellet when Tris was added.

Refolding Protein

Spectra were taken to monitor how the protein was affected by the changing pH (the protein was diluted as a result of adding the buffer). A slight shift in peak was seen when the pH was increased. A spectrum was also taken of the solution in which the protein fibers were made to see if there were any gold nanoparticles in the solution.


Spectra 111511.png

Notes