User:Eleni N. Kalivas/Notebook/CHEM-571/2013/11/19

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Current revision (14:07, 10 December 2013) (view source)
(Objective)
 
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==Objective==
==Objective==
 +
*Prepare AuNP-protein solutions for biomineralization
 +
*Analyze AuNP-protein solutions prepared on 10/30/2013using AA spectroscopy.
 +
<br>
 +
 +
==AuNP solution preparation==
 +
*The general protocol for the preparation of AuNPs described on 08/28/2013 was followed.
 +
*20 AuNP solutions were prepared with BSA and Invertase proteins.
 +
<br>
 +
Table 1. Gold Standard Solution preparation
 +
<br>
 +
{| {{table}}
 +
| align="center" style="background:#f0f0f0;"|''' Preparation of Gold Stock Solution'''
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| align="center" style="background:#f0f0f0;"|'''''''''
 +
|-
 +
|Molecular Weight (g/mol)||393.83
 +
|-
 +
| mass Au(g)||0.0354
 +
|-
 +
| volume stock(L)||0.025
 +
|-
 +
| Concentration (M)||0.00359546
 +
|}
 +
<br>
 +
Table 2. Preparation of BSA stock solution
 +
<br>
 +
{| {{table}}
 +
| align="center" style="background:#f0f0f0;"|'''Preparation of BSA stock solution'''
 +
| align="center" style="background:#f0f0f0;"|''''''
 +
|-
 +
|Molecular weight (g/mol)||66000
 +
|-
 +
| mass (g)||0.0432
 +
|-
 +
| volume stock (L)||0.03
 +
|-
 +
| Concentration (M)||2.18182E-05
 +
|}
 +
<br>
 +
Table 3. Preparation of Invertase Stock solution
 +
<br>
 +
{| {{table}}
 +
| align="center" style="background:#f0f0f0;"|'''Preparation Invertase Stock'''
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| align="center" style="background:#f0f0f0;"|''''''
 +
|-
 +
| Molecular weight (g/mol)||135000
 +
|-
 +
| mass invertase (g)||0.1015
 +
|-
 +
| volume stock (L)||0.025
 +
|-
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| concentration (M)||3.00741E-05
 +
|}
 +
<br>
 +
Table 4. Preparation of BSA-AuNP solutions
 +
<br>
 +
{| {{table}}
 +
| align="center" style="background:#f0f0f0;"|'''Ratio'''
 +
| align="center" style="background:#f0f0f0;"|'''Amount of gold added(mL)'''
 +
| align="center" style="background:#f0f0f0;"|'''concentration BSA (M)'''
 +
| align="center" style="background:#f0f0f0;"|'''volume BSA (mL)'''
 +
| align="center" style="background:#f0f0f0;"|'''volume Water added (mL)'''
 +
|-
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| 105||0.347660664||2.38095E-06||0.545634921||4.106704416
 +
|-
 +
| 110||0.347660664||2.27273E-06||0.520833333||4.131506003
 +
|-
 +
| 115||0.347660664||2.17391E-06||0.498188406||4.15415093
 +
|-
 +
| 120||0.347660664||2.08333E-06||0.477430556||4.174908781
 +
|-
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| 125||0.347660664||0.000002||0.458333333||4.194006003
 +
|-
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| 130||0.347660664||1.92308E-06||0.440705128||4.211634208
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|-
 +
| 135||0.347660664||1.85185E-06||0.424382716||4.22795662
 +
|-
 +
| 140||0.347660664||1.78571E-06||0.40922619||4.243113146
 +
|-
 +
| 145||0.347660664||1.72414E-06||0.395114943||4.257224394
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|-
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| 150||0.347660664||1.66667E-06||0.381944444||4.270394892
 +
|}
 +
<br>
 +
Table 5. Preparation of Invertase-AuNP solutions
 +
<br>
 +
{| {{table}}
 +
| align="center" style="background:#f0f0f0;"|'''Ratio'''
 +
| align="center" style="background:#f0f0f0;"|'''Amount gold added (mL)'''
 +
| align="center" style="background:#f0f0f0;"|'''Concentration Invertase (M)'''
 +
| align="center" style="background:#f0f0f0;"|'''Volume Invertase (mL)'''
 +
| align="center" style="background:#f0f0f0;"|'''Volume water added (mL)'''
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|-
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| 30||0.347660664||8.33333E-06||1.38546798||3.266871356
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|-
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| 50||0.347660664||0.000005||0.831280788||3.821058548
 +
|-
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| 70||0.347660664||3.57143E-06||0.593771992||4.058567345
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|-
 +
| 90||0.347660664||2.77778E-06||0.46182266||4.190516676
 +
|-
 +
| 110||0.347660664||2.27273E-06||0.377854904||4.274484432
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|-
 +
| 130||0.347660664||1.92308E-06||0.31972338||4.332615956
 +
|-
 +
| 150||0.347660664||1.66667E-06||0.277093596||4.37524574
 +
|-
 +
| 170||0.347660664||1.47059E-06||0.244494349||4.407844987
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|-
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| 190||0.347660664||1.31579E-06||0.218758102||4.433581234
 +
|-
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| 210||0.347660664||1.19048E-06||0.197923997||4.454415339
 +
|}
 +
<br>
 +
 +
 +
==AA spectroscopy==
 +
<br>
 +
'''General Protocol'''
 +
#Transfer 3mL of sample from test tubes into a 10mL falcon tube.
 +
#Centrifuge 3mL protein-AuNP solutions for 30 minutes at 4000rpm. Be sure that the centrifuge is balanced.
 +
#Prepare 5 standard Au solutions (10μg/mL, 20μg/mL, 30μg/mL, 40μg/mL, 50μg/mL) following protocol described on [[User:Moira M. Esson/Notebook/CHEM-571/2013/09/10|09/10/2013]].
 +
#Run samples on AA.
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<br>
 +
Table 6. Standard Au Solution preparation from 1000μg/mL AA Au gold standard solution
 +
<br>
 +
{| {{table}}
 +
| align="center" style="background:#f0f0f0;"|'''Concentration(μg/mL)'''
 +
| align="center" style="background:#f0f0f0;"|'''Volume of stock Au solution(mL)'''
 +
| align="center" style="background:#f0f0f0;"|'''Volume Deionized water added(mL)'''
 +
|-
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| 10||0.1||9.9
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|-
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| 20||0.2||9.8
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|-
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| 30||0.3||9.7
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|-
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| 40||0.4||9.6
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|-
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| 50||0.5||9.5
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|}
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<br>
 +
Figure 1. Calibration curve of gold standard solutions
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<br>
 +
[[Image:Calibration Curve of Gold Standards zem11192013 .png]]
 +
<br>
 +
Figure 2. Atomic Absorbance Spectra of Myoglobin:AuNP solutions
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<br>
 +
[[Image:Atomic Absorbance Spectra of Myoglobin AuNP solutions zem11192013.png]]
 +
<br>
 +
Figure 3. Atomic Absorbance Spectra of Lysozyem:AuNP solutions
 +
<br>
 +
[[Image:Atomic Absorbance Spectra of Lysozyme AuNP solutions zem 11192013 .png]]
 +
<br>
 +
*The only solution that was colored was the 30 ratio. All other solutions appeared colorless.
 +
Figure 4. Absorbance vs. Concentration plot of Myoglobin:AuNP solutions
 +
<br>
 +
[[Image:Absorbance vs. Concentration plot of Myoglobin-AuNP solutions zem11192013.png]]
 +
<br>
 +
Figure 5. Absorbance vs. Concentration plot of AuNP-Lysozyme Solutions
 +
<br>
 +
[[Image:Absorbance vs. Concentration plot of Lysozyme-AuNP solutions zem11192013.png]]
 +
 +
 +

Current revision

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Objective

  • Prepare AuNP-protein solutions for biomineralization
  • Analyze AuNP-protein solutions prepared on 10/30/2013using AA spectroscopy.


AuNP solution preparation

  • The general protocol for the preparation of AuNPs described on 08/28/2013 was followed.
  • 20 AuNP solutions were prepared with BSA and Invertase proteins.


Table 1. Gold Standard Solution preparation

Preparation of Gold Stock Solution ''''
Molecular Weight (g/mol)393.83
mass Au(g)0.0354
volume stock(L)0.025
Concentration (M)0.00359546


Table 2. Preparation of BSA stock solution

Preparation of BSA stock solution '
Molecular weight (g/mol)66000
mass (g)0.0432
volume stock (L)0.03
Concentration (M)2.18182E-05


Table 3. Preparation of Invertase Stock solution

Preparation Invertase Stock '
Molecular weight (g/mol)135000
mass invertase (g)0.1015
volume stock (L)0.025
concentration (M)3.00741E-05


Table 4. Preparation of BSA-AuNP solutions

Ratio Amount of gold added(mL) concentration BSA (M) volume BSA (mL) volume Water added (mL)
1050.3476606642.38095E-060.5456349214.106704416
1100.3476606642.27273E-060.5208333334.131506003
1150.3476606642.17391E-060.4981884064.15415093
1200.3476606642.08333E-060.4774305564.174908781
1250.3476606640.0000020.4583333334.194006003
1300.3476606641.92308E-060.4407051284.211634208
1350.3476606641.85185E-060.4243827164.22795662
1400.3476606641.78571E-060.409226194.243113146
1450.3476606641.72414E-060.3951149434.257224394
1500.3476606641.66667E-060.3819444444.270394892


Table 5. Preparation of Invertase-AuNP solutions

Ratio Amount gold added (mL) Concentration Invertase (M) Volume Invertase (mL) Volume water added (mL)
300.3476606648.33333E-061.385467983.266871356
500.3476606640.0000050.8312807883.821058548
700.3476606643.57143E-060.5937719924.058567345
900.3476606642.77778E-060.461822664.190516676
1100.3476606642.27273E-060.3778549044.274484432
1300.3476606641.92308E-060.319723384.332615956
1500.3476606641.66667E-060.2770935964.37524574
1700.3476606641.47059E-060.2444943494.407844987
1900.3476606641.31579E-060.2187581024.433581234
2100.3476606641.19048E-060.1979239974.454415339



AA spectroscopy


General Protocol

  1. Transfer 3mL of sample from test tubes into a 10mL falcon tube.
  2. Centrifuge 3mL protein-AuNP solutions for 30 minutes at 4000rpm. Be sure that the centrifuge is balanced.
  3. Prepare 5 standard Au solutions (10μg/mL, 20μg/mL, 30μg/mL, 40μg/mL, 50μg/mL) following protocol described on 09/10/2013.
  4. Run samples on AA.


Table 6. Standard Au Solution preparation from 1000μg/mL AA Au gold standard solution

Concentration(μg/mL) Volume of stock Au solution(mL) Volume Deionized water added(mL)
100.19.9
200.29.8
300.39.7
400.49.6
500.59.5


Figure 1. Calibration curve of gold standard solutions
Image:Calibration Curve of Gold Standards zem11192013 .png
Figure 2. Atomic Absorbance Spectra of Myoglobin:AuNP solutions
Image:Atomic Absorbance Spectra of Myoglobin AuNP solutions zem11192013.png
Figure 3. Atomic Absorbance Spectra of Lysozyem:AuNP solutions
Image:Atomic Absorbance Spectra of Lysozyme AuNP solutions zem 11192013 .png

  • The only solution that was colored was the 30 ratio. All other solutions appeared colorless.

Figure 4. Absorbance vs. Concentration plot of Myoglobin:AuNP solutions
Image:Absorbance vs. Concentration plot of Myoglobin-AuNP solutions zem11192013.png
Figure 5. Absorbance vs. Concentration plot of AuNP-Lysozyme Solutions
Image:Absorbance vs. Concentration plot of Lysozyme-AuNP solutions zem11192013.png




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