Difference between revisions of "User:Michael F. Nagle/Notebook/Chem 571/2012/11/07"

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| colspan="2"|
 
| colspan="2"|
 
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 +
==Objective==
 +
*Determine concentration of ADA prepared [[User:Michael_F._Nagle/Notebook/Chem_571/2012/10/23|Oct. 23]] via Bradford Assay
 
==Bradford Assay==
 
==Bradford Assay==
  
A standard curve was obtained with the following concentrations of BSA:
+
*Solutions were prepared with 715μL 1x Bio-Rad Bradford reagant and the following concentrations of BSA:
  
[table]
+
{| {{table}}
[tr]
+
| align="center" style="background:#f0f0f0;"|'''volume BSA (uL)'''
[th]volume BSA (uL)[/th]
+
| align="center" style="background:#f0f0f0;"|'''volume H2O (uL'''
[th]volume H2O (uL)[/th]
+
| align="center" style="background:#f0f0f0;"|'''[BSA] (mg/mL)'''
[th]concentration BSA (mg/mL)[/th]
+
| align="center" style="background:#f0f0f0;"|'''[BSA] (M)'''
[th]concentration BSA (molarity)[/th]
+
|-
[/tr]
+
| 0||30||0||0.00E+00
[tr]
+
|-
[td]0[/td]
+
| 5||25||0.24||3.61E-06
[td]30[/td]
+
|-
[td]0[/td]
+
| 10||20||0.15||2.26E-06
[td]0.00E+00[/td]
+
|-
[/tr]
+
| 15||15||0.13||2.01E-06
[tr]
+
|-
[td]5[/td]
+
| 20||10||0.15||2.26E-06
[td]25[/td]
+
|-
[td].24[/td]
+
| 25||5||0.24||3.61E-06
[td]3.61E-06[/td]
+
|-
[/tr]
+
| 30||0||1||1.51E-05
[tr]
+
|-
[td]10[/td]
+
|}
[td]20[/td]
 
[td].15[/td]
 
[td]2.26E-06[/td]
 
[/tr]
 
[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]
 
 
<Br>
 
<Br>
*The ADA made [[User:Michael_F._Nagle/Notebook/Chem_571/2012/10/23|Oct. 23]] was analyzed.
+
*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.
+
*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.
 +
 
 +
==Data==
 +
[[Image:Bradforassay.png]]
 +
{| {{table}}
 +
| align="center" style="background:#f0f0f0;"|'''Fraction #'''
 +
| align="center" style="background:#f0f0f0;"|'''Absorbance'''
 +
| align="center" style="background:#f0f0f0;"|'''[ADA] [mg/mL]'''
 +
| align="center" style="background:#f0f0f0;"|'''molar absorptivity [mL/mg*cm-1]'''
 +
| 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
 +
|-
 +
| 9||0.819||0.0137||59.7||65.0
 +
|-
 +
|
 +
|}
 +
<br>Frac 2 was found to be 42µM, while 8 was 54.4µM and 9 was 65µM.
 +
 
 
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|}
 
|}
  
 
__NOTOC__
 
__NOTOC__

Revision as of 03:51, 7 December 2012

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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)
0 30 0 0.00E+00
5 25 0.24 3.61E-06
10 20 0.15 2.26E-06
15 15 0.13 2.01E-06
20 10 0.15 2.26E-06
25 5 0.24 3.61E-06
30 0 1 1.51E-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

Bradforassay.png

Fraction # Absorbance [ADA] [mg/mL] molar absorptivity [mL/mg*cm-1] Molarity (µM)
2 0.93 0.0177 52.4 42.0
8 0.757 0.0115 65.9 54.4
9 0.819 0.0137 59.7 65.0


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