User:Puja Mody/Notebook/Chem 571: Gold Nanoparticles/2012/10/02: Difference between revisions

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==FPLC==


==Horseradish Peroxidase Assay Absorbance==


==Goals==
==Goals==
Run an FPLC of the protein in order to purify it.
* Run a  HPR assay that varies the concentration of Gold nanoparticle solution (AuNP/BSa)


==Procedure==
==Procedure==
*FPLC is Fast protein liquid chromatography and is used in order to purify the protein and uses a system of pumps in order to push the various liquids around the apparatus.  
* We followed the HRP Protocol  [[Image:3760-Protocol HRP AbsorbanceAssay.pdf]] and pay particular attention to what the other group( [[Image:HRP Assays.pdf]] had done two weeks ago in regards to this same experiment.   
*First, the system needed to be equilibrated. The superloop pushed proteins through the system and can hold 50 mL. In A<sub>1</sub> the tube was placed in the binding buffer and for B<sub>1</sub> the tube was placed in the elution buffer in order to equilibrate the system.
* The previous group had already tested to see how varying the concentrations of aminoantipyrine and hydrogen perioxide had affected the rate of the reaction after the addition of the HRP. As a result, we wanted to rest what effect the addition of the gold nanoparticles had on that reaction.  
* We needed to run 25mL of Buffer through the column at a pressure of .14/.15 ( the pressure tells if there is anything clogging the system)
** The conductivity meter reads how much salt is in the system
** Imidizole also contains the functional group of histidine.
* We needed to first run the binding buffer, followed by the elution buffer, and then the binding buffer once more through the column otherwise the column will have too much imidazole.
* The column only run at 5mL/minute. The nickel column of the instrument can hold 5mL and contains polymer beads. The ADA protein contains specifically a histag on the protein of 6 histidines (which is the amount necessary in order to have a night enough affinity to nickel). Histidine has an affinity to nickel and therefore will bind with it.  


* We then put the protein on. We ran sample 1 first.  
# We made .2 M sodium phosphate at a pH of 7 ( we made 60mL)
* The protein histidines (in the ADA) bind with the nickel in the column. To get the protein out, we use the elution buffer because the imidazole histidines compete with the histidine in the column and binds to the nickel. This results in the protein being pushed out.  
#0.0017 M of hydrogen peroxide was made by adding 1 mL of 30 % hydrogen peroxide to 100 ml
distilled water. It was then further diluted by taking 1mL of the solution in 50mL of 0.2 M
sodium phosphate buffer.
#0.0025 M 4-Aminoantipyrine with 0.17 M phenol:
** Because the iodophenol was immiscible in water, it had to be dissolved in DMSO. It was dissolved in 1mL of DMSO to make a concentration of 700μM. The 25mg of aminoantipyrine was dissolved in 50mL of water.  
# 1mg of HRP was dissolved in 1mL of distilled water.  


Based on the other groups tests, we decided to keep all our variables constant except for the percent by volume of the AuNP added into each cuvet.The Gold Nanoparticle were made in a 1% citrate solution rather than in BSa.  We decided that between the two separate trials, we would vary the concentration of the AAP because on the other groups results, it seems that the concentrations 156.25 mM and 312 mM were the most linear.
Because of changes in calculations and incorrect data results from the UV-Vis instrument, The spectra that we run today did not react in the manner in which we were looking for. However, please look at [[User:Mary Mendoza/Notebook/CHEM 571 Experimental Biological Chemistry I/2012/10/02 |Mary's Notebook]] to see the spectras and analysis collected on this day.




Revision as of 11:55, 23 October 2012

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Horseradish Peroxidase Assay Absorbance

Goals

  • Run a HPR assay that varies the concentration of Gold nanoparticle solution (AuNP/BSa)

Procedure

  • We followed the HRP Protocol File:3760-Protocol HRP AbsorbanceAssay.pdf and pay particular attention to what the other group( File:HRP Assays.pdf had done two weeks ago in regards to this same experiment.
  • The previous group had already tested to see how varying the concentrations of aminoantipyrine and hydrogen perioxide had affected the rate of the reaction after the addition of the HRP. As a result, we wanted to rest what effect the addition of the gold nanoparticles had on that reaction.
  1. We made .2 M sodium phosphate at a pH of 7 ( we made 60mL)
  2. 0.0017 M of hydrogen peroxide was made by adding 1 mL of 30 % hydrogen peroxide to 100 ml

distilled water. It was then further diluted by taking 1mL of the solution in 50mL of 0.2 M sodium phosphate buffer.

  1. 0.0025 M 4-Aminoantipyrine with 0.17 M phenol:
    • Because the iodophenol was immiscible in water, it had to be dissolved in DMSO. It was dissolved in 1mL of DMSO to make a concentration of 700μM. The 25mg of aminoantipyrine was dissolved in 50mL of water.
  1. 1mg of HRP was dissolved in 1mL of distilled water.

Based on the other groups tests, we decided to keep all our variables constant except for the percent by volume of the AuNP added into each cuvet.The Gold Nanoparticle were made in a 1% citrate solution rather than in BSa. We decided that between the two separate trials, we would vary the concentration of the AAP because on the other groups results, it seems that the concentrations 156.25 mM and 312 mM were the most linear.

Because of changes in calculations and incorrect data results from the UV-Vis instrument, The spectra that we run today did not react in the manner in which we were looking for. However, please look at Mary's Notebook to see the spectras and analysis collected on this day.



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