User:Khyra A. Neal/Notebook/Chem 571/2014/10/29: Difference between revisions

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(Autocreate 2014/10/29 Entry for User:Khyra_A._Neal/Notebook/Chem_571)
 
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==Entry title==
==October 29, 2014==
* Insert content here...


===Tasklist===


1.  Extract Lysozyme and CaCl<sub>2</sub> solutions
* Solutions were prepared on [http://openwetware.org/wiki/User:Khyra_A._Neal/Notebook/Chem_571/2014/10/28 October 28, 2014]
* Extracted with a pasteur pipette and transferred to 30 mL extraction vials
2.  Bradford Analysis
* Extracted Lysozyme solutions that were dialyzed against CaCl<sub>2</sub>
* 200 μL Bradford Reagent (diluted 1:3)
* 50 μL Sample Solution
* 750 μL 50 mM Tris/ 50 mM NaCl Buffer
* Run UV-Vis between 400 nm and 800 nm
[[Image:Bradford_of_Lys_vs_CaCl2.jpg]]
3. Ca<sup>2+</sup> ISE
<u>Ca2+ ISE</u>
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''CaCl<sub>2</sub> concentration'''
| align="center" style="background:#f0f0f0;"|'''mV '''
|-
| 5μM CaCl<sub>2</sub> || 11.8
|-
| Lys (1 ) || 4.6
|-
| 50μM CaCl<sub>2</sub> || 4.2
|-
| Lys (2) || 0.1
|-
| 500μM CaCl<sub>2</sub> || 27.4
|-
| Lys (3) || 24.4
|-
| 5mM CaCl<sub>2</sub> || 52.1
|-
| Lys (4) || 51.3
|-
| 50mM CaCl<sub>2</sub>|| 77.3
|-
| Lys (5) || 77.0
|-
|}
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|}
|}
5. UV-Vis and Fluorescence
* Sample Solutions were diluted 1:100
* Transfer 100 μL of sample solution to a small volume cuvette
* Measure absorbance between 200 nm and 800 nm
* Use SDS, HCl, HPLC, & methanol to clean cuvette after each use
* Fluorescence spectra was obtained to determine how binding of the protein was affected


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Revision as of 16:00, 30 October 2014

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October 29, 2014

Tasklist

1. Extract Lysozyme and CaCl2 solutions

  • Solutions were prepared on October 28, 2014
  • Extracted with a pasteur pipette and transferred to 30 mL extraction vials

2. Bradford Analysis

  • Extracted Lysozyme solutions that were dialyzed against CaCl2
  • 200 μL Bradford Reagent (diluted 1:3)
  • 50 μL Sample Solution
  • 750 μL 50 mM Tris/ 50 mM NaCl Buffer
  • Run UV-Vis between 400 nm and 800 nm

3. Ca2+ ISE

Ca2+ ISE

CaCl2 concentration mV
5μM CaCl2 11.8
Lys (1 ) 4.6
50μM CaCl2 4.2
Lys (2) 0.1
500μM CaCl2 27.4
Lys (3) 24.4
5mM CaCl2 52.1
Lys (4) 51.3
50mM CaCl2 77.3
Lys (5) 77.0

5. UV-Vis and Fluorescence

  • Sample Solutions were diluted 1:100
  • Transfer 100 μL of sample solution to a small volume cuvette
  • Measure absorbance between 200 nm and 800 nm
  • Use SDS, HCl, HPLC, & methanol to clean cuvette after each use
  • Fluorescence spectra was obtained to determine how binding of the protein was affected



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