User:Michael F. Nagle/Notebook/Chem 571/2012/11/07

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Current revision (06:51, 7 December 2012) (view source)
(Data)
 
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==Objective==
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*Determine concentration of ADA prepared [[User:Michael_F._Nagle/Notebook/Chem_571/2012/10/23|Oct. 23]] via Bradford Assay
==Bradford Assay==
==Bradford Assay==
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A standard curve was obtained with the following concentrations of BSA:
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*Solutions were prepared with 715μL 1x Bio-Rad Bradford reagant and the following concentrations of BSA:
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[table]
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{| {{table}}
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[tr]
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| align="center" style="background:#f0f0f0;"|'''volume BSA (uL)'''
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[th]volume BSA (uL)[/th]
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| align="center" style="background:#f0f0f0;"|'''volume H2O (uL'''
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[th]volume H2O (uL)[/th]
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| align="center" style="background:#f0f0f0;"|'''[BSA] (mg/mL)'''
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[th]concentration BSA (mg/mL)[/th]
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| align="center" style="background:#f0f0f0;"|'''[BSA] (M)'''
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[th]concentration BSA (molarity)[/th]
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|-
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[/tr]
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| 0||30||0||0.00E+00
-
[tr]
+
|-
-
[td]0[/td]
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| 5||25||0.24||3.61E-06
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[td]30[/td]
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|-
-
[td]0[/td]
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| 10||20||0.15||2.26E-06
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[td]0.00E+00[/td]
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|-
-
[/tr]
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| 15||15||0.13||2.01E-06
-
[tr]
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|-
-
[td]5[/td]
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| 20||10||0.15||2.26E-06
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[td]25[/td]
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|-
-
[td].24[/td]
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| 25||5||0.24||3.61E-06
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[td]3.61E-06[/td]
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|-
-
[/tr]
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| 30||0||1||1.51E-05
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[tr]
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|-
-
[td]10[/td]
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|}
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[td]20[/td]
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-
[td].15[/td]
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-
[td]2.26E-06[/td]
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-
[/tr]
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-
[tr]
+
-
[td]15[/td]
+
-
[td]15[/td]
+
-
[td].13[/td]
+
-
[td]2.01E-06[/td]
+
-
[/tr]
+
-
[tr]
+
-
[td]20[/td]
+
-
[td]10[/td]
+
-
[td].15[/td]
+
-
[td]2.26E-06[/td]
+
-
[/tr]
+
-
[tr]
+
-
[td]25[/td]
+
-
[td]5[/td]
+
-
[td].24[/td]
+
-
[td]3.61E-06[/td]
+
-
[/tr]
+
-
[tr]
+
-
[td]30[/td]
+
-
[td]0[/td]
+
-
[td]1[/td]
+
-
[td]1.51E-05[/td]
+
-
[/tr]
+
-
[/table]
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<Br>
<Br>
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*The ADA made [[User:Michael_F._Nagle/Notebook/Chem_571/2012/10/23|Oct. 23]] was analyzed.
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*A UV/Vis calibration curve was obtained by analyzing the samples and plotting BSA concentration against absorbance at 595nm.
-
*The ADA's peak was compared to those of BSA's linear regression in order to determine concentration.
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*The [[User:Michael_F._Nagle/Notebook/Chem_571/2012/11/06|ADA purified yesterday]], labelled Frac 9, was analyzed along with Fracs 2 and 8 purified [[User:Michael_F._Nagle/Notebook/Chem_571/2012/09/26|9/26]] via UV/Vis and it's peak was compared to the linear regression of BSA's in order to determine concentration.
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==Data==
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[[Image:Bradforassay.png]]
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{| {{table}}
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| align="center" style="background:#f0f0f0;"|'''Fraction #'''
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| align="center" style="background:#f0f0f0;"|'''Absorbance'''
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| align="center" style="background:#f0f0f0;"|'''[ADA] [mg/mL]'''
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| align="center" style="background:#f0f0f0;"|'''molar absorptivity [mL/mg*cm-1]'''
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| align="center" style="background:#f0f0f0;"|'''Molarity (µM)'''
 +
|-
 +
| 2||0.93||0.0177||52.4||42.0
 +
|-
 +
| 8||0.757||0.0115||65.9||54.4
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|-
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| 9||0.819||0.0137||59.7||65.0
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|-
 +
|
 +
|}
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<br>Frac 2 was found to be 42µM, while 8 was 54.4µM and 9 was 65µM.
 +
 
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__NOTOC__
__NOTOC__

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Objective

  • Determine concentration of ADA prepared Oct. 23 via Bradford Assay

Bradford Assay

  • Solutions were prepared with 715μL 1x Bio-Rad Bradford reagant and the following concentrations of BSA:
volume BSA (uL) volume H2O (uL [BSA] (mg/mL) [BSA] (M)
03000.00E+00
5250.243.61E-06
10200.152.26E-06
15150.132.01E-06
20100.152.26E-06
2550.243.61E-06
30011.51E-05


  • A UV/Vis calibration curve was obtained by analyzing the samples and plotting BSA concentration against absorbance at 595nm.
  • The ADA purified yesterday, labelled Frac 9, was analyzed along with Fracs 2 and 8 purified 9/26 via UV/Vis and it's peak was compared to the linear regression of BSA's in order to determine concentration.

Data

Image:Bradforassay.png

Fraction # Absorbance [ADA] [mg/mL] molar absorptivity [mL/mg*cm-1] Molarity (µM)
20.930.017752.442.0
80.7570.011565.954.4
90.8190.013759.765.0


Frac 2 was found to be 42µM, while 8 was 54.4µM and 9 was 65µM.


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