User:Zachary I. Mendel/Notebook/Zacks Notebook/2014/02/18: Difference between revisions

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*one solution was 10^-7M protein in nanoparticles. The other was 10^-8
*one solution was 10^-7M protein in nanoparticles. The other was 10^-8
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measure activity of HRP nanoparticles
#HRP-AuNP complex solution from the alpha method and the polydopamine-coated AuNPs were placed in high velocity tubes.
#The nano particle solutions were spun at 18,000rpm for 3 hours.
#The supernatants were discarded and the pellets were resuspended in water.
#The polydopamine-coated AuNPs were stored at 4 degrees Celsius
#The resuspended HRP-AuNP complex solutions were analyzed for activity using UV-Vis
##The nano particles were added to hydrogen peroxide and OPD (Ophenylenediamine)
##Active HRP catalyzes the reaction of Ophenylenediamine with H2O2 to produce 2,3-Diaminophenazine
[[Image:Screen Shot 2014-02-16 at 10.44.57 PM.png|400px]]
###Stock solution of OPD with a concentration of 1mg/mL
###Stock solution of H<sub>2</sub>O<sub>2</sub> 113uL


==Notes==
==Notes==

Revision as of 11:32, 25 February 2014

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February 18th

Goals

  • Make graph showing max peak over time
  • Making HRP nanoparticles using dopamine method
  • Run UV vis on HRP nanoparticles to see if there is any difference after having it run a week later
  • Run UV vis on HRP supernatant
    • We want to see there is free HRP in the solution and that all of the HRP did not get spun down



  • make hemoglobin nanoparticles using the alpha method

Procedures

Other things to do

  • take aliquot of 10^-8M HRP nanoparticles and put in freezer



  • Protein Gold Nanoparticles
    • I already have my 10^-6M Hemoglobin solution made
    • Take 1ml of the solution and add to 9ml of nanoparticles...take 100ul and add to 9.9mls of gold to make solutions


  • one solution was 10^-7M protein in nanoparticles. The other was 10^-8M.


Making HRP nanoparticles

    • First I made the buffer solution. The pH was 5. I made it using 0.0003 moles of acetic acid 1M and 0.0006 moles of potassium acetate
      • add 300ul of acid and 0.059g of base
    • Going to make 10^-6M HRP solution in the buffer
      • HRP molecular mass is 44kDa or 44000g/mol
        • added 0.0044g of HRP
      • we then filled up 100ml volumetric flask
    • Take 1ml of the solution and add to 9ml of nanoparticles...take 100ul and add to 9.9mls of gold to make solutions


  • one solution was 10^-7M protein in nanoparticles. The other was 10^-8



measure activity of HRP nanoparticles

  1. HRP-AuNP complex solution from the alpha method and the polydopamine-coated AuNPs were placed in high velocity tubes.
  2. The nano particle solutions were spun at 18,000rpm for 3 hours.
  3. The supernatants were discarded and the pellets were resuspended in water.
  4. The polydopamine-coated AuNPs were stored at 4 degrees Celsius
  5. The resuspended HRP-AuNP complex solutions were analyzed for activity using UV-Vis
    1. The nano particles were added to hydrogen peroxide and OPD (Ophenylenediamine)
    2. Active HRP catalyzes the reaction of Ophenylenediamine with H2O2 to produce 2,3-Diaminophenazine

      1. Stock solution of OPD with a concentration of 1mg/mL
      2. Stock solution of H2O2 113uL

Notes

  • HRP was 10^-8 M added to nanoparticles


  • when I made the new HRP particles, there was definitely some wierd impurities present...this is important to note if the HRP doesn't work

Reflections


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