User:Moira M. Esson/Notebook/CHEM-571/2013/10/16

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(Catalytic activity testing)
(UV/vis of Citrate and BSA samples)
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==UV/vis of Citrate and BSA samples==
==UV/vis of Citrate and BSA samples==
*A new BSA-Au sample was prepared because the protein had crashed out of solution. The BSA solution was prepared following the protocol described on [[User:Moira M. Esson/Notebook/CHEM-571/2013/08/28|08/28/2013]]. The absorbance of the BSA solution will be determined during the next meeting.
*A new BSA-Au sample was prepared because the protein had crashed out of solution. The BSA solution was prepared following the protocol described on [[User:Moira M. Esson/Notebook/CHEM-571/2013/08/28|08/28/2013]]. The absorbance of the BSA solution will be determined during the next meeting.
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  Preparation of BSA stock solution
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  0.025g BSA in 25mL tris buffer(described above)-->15μM BSA
*A 1/10 dilution of Citrate-Au solution was measured using UV/vis
*A 1/10 dilution of Citrate-Au solution was measured using UV/vis
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Figure 2. Absorbance spectra of 1/10 dilution of Citrate-AuNP
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Figure 2. Absorbance spectra of 1/10 dilution of Citrate-AuNP and 1/10 dilution of 30:1 HRP-Au solution
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[[Image:HRP and Citrate AuNP.png|750px]]
[[Image:HRP and Citrate AuNP.png|750px]]

Revision as of 16:06, 21 October 2013

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Objectives

  • Test the enzymatic/catalytic activity of HRP-AuNPs using luminol and UV/vis with the goal of determining the activity of HRP-AuNPs.
  • Calculate the absorbance prepared Citrate-Au solutions and BSA-Au solutions as standards (solutions were previously prepared on 08/28/2013


Catalytic activity testing

  • Catalytic activity was tested using UV/vis spectroscopy.
  • 3mL sample of luminol, H2O2 and buffer was placed in cuvette with micro-stir bar. After 1min., 100μL HRP-Au was added.
  • A spectrum was taken every 15seconds for twenty minutes.


Sample preparation

 Buffer:0.6140g Tris in 1L, pH set to 8 with HCl ---> 5.1mM
 Luminol:Dissolve 12.9mg luminol in 300uL of DMSO and add to 50mL buffer---> 1.46mM
 H2O2: 312uL 30% H2O2 into 100mL buffer ---> Should be 45mM


  • Preparation of HRP-AuNP samples:
    • General Description of the samples:
      • The final concentration of luminol is such that the absorbance at 333nm is less than one(this was confirmed using UV/vis)
      • The final concentration of hydrogen peroxide is fifty times that of the luminol.
      • Using the 60:1 [Au:HRP] solution, 100uL will be used
      • Add buffer so that the final concentration is 3mL
 Calculations for preparation of sample:
   Luminol stock solution preparation 5mL 7.26E-5 M luminol: 0.261mL luminol stock added to 5mL buffer.
   H2O2 stock preparation of 5mL 0.0038M(this is 50x more concentrated): 0.169mL stock in 5mL buffer.
  • Final 3mL solution of luminol, H2O2 and buffer was prepared by adding 1mL luminol, 1mL H2O2 and 1mL buffer.
  • Concentration of luminol was chosen based on previous luminol data on 10/01/2013.


Testing of HRP-AuNP activity

  • The first prepared sample of luminol and H2O2 already had completely oxidized luminol. A new solution was prepared.
  • No activity of the HRP was observed (see figure below).


UV/vis of Citrate and BSA samples

  • A new BSA-Au sample was prepared because the protein had crashed out of solution. The BSA solution was prepared following the protocol described on 08/28/2013. The absorbance of the BSA solution will be determined during the next meeting.
 Preparation of BSA stock solution
  0.025g BSA in 25mL tris buffer(described above)-->15μM BSA
  • A 1/10 dilution of Citrate-Au solution was measured using UV/vis


Figure 2. Absorbance spectra of 1/10 dilution of Citrate-AuNP and 1/10 dilution of 30:1 HRP-Au solution




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