User:Daniel-Mario Larco/Notebook/AU Biodesign Lab - 09/03/2013/2014/03/04

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Gamma Method Nanoparticles

  • Today we will synthesize protein coated AuNPs using the gamma method (reactant free nano particles)
  • Because we have a limited amount of these nano particles, we prepared a 1/10 dilution by mixing 3mL of the nano particle solution with 27mL of water.
  • This dilution brought up a question of concentration
  1. For this we ran UV-Vis of the citrate nanopaticle solutions used in the other methods to determine their AuNP concentration
    1. The data was analyzed using thisthis reference
    2. The concentration was determined to about 10^12
  1. In order to synthesize the protein coated AuNPs, we used the same ratio as the other methods
  2. The protein stocks were prepared by dissolving protein in 50mM Tris buffer with pH adjusted to be as close to the protein's PI as possible
  3. The final concentration of the protein stocks was about 0.1mg per mL
  4. just as in the two other methods, a 1:10 and 1:100 AuNP to protein volume ratio were prepared
    1. 1mL protein in 9mL solution and 100uL in 9.9mL solution
  5. The solutions were left to incubate at room temperature for 30 minutes
  6. the solutions were then placed in the centrifuge for 30 minutes at 6500G (~7,400rpm)
  7. the supernatant and pellet were separated and the pellet resuspended in tris buffer
  8. Both sets of solutions were placed in the refrigerator until ready for testing

-  Because these nano particles were 100X less concentrated than the AuNPs used for the other methods. The activity is expected to be lower in this method

because less protein will bind.