User:Zachary I. Mendel/Notebook/Zacks Notebook/2014/02/18: Difference between revisions
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== | ==February 18th== | ||
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==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 | |||
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*make hemoglobin nanoparticles using the alpha method | |||
==Procedures== | |||
Other things to do | |||
*take aliquot of 10^-8M HRP nanoparticles and put in freezer | |||
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*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 | |||
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*one solution was 10^-7M protein in nanoparticles. The other was 10^-8M. | |||
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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 | |||
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*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== | |||
*HRP was 10^-8 M added to nanoparticles | |||
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*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 | |||
==Data== | |||
Alpha HRP Pellet | |||
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[[Image:HRP AUnP Pellet.png|Alpha HRP Pellet]] | |||
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This graph shows absorbance increases in the HRP Pellet over time | |||
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Alpha HRP supernatant | |||
[[Image:HRP Supernatant.png]] | |||
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This graph shows absorbance increasing slightly in the HRP supernatant over time | |||
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Control. OPD and H202 only | |||
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[[Image:CONTROL HRP ALPHA.png]] | |||
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This graph reveals what happens to absorbance over time when no HRP nanoparticles are present | |||
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Here there is only hydrogen peroxide and OPD | |||
==Reflections== | |||
Revision as of 11:43, 17 April 2014
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February 18thGoals
ProceduresOther things to do
Notes
DataAlpha HRP Pellet
Reflections |