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==Entry title==
 
* Insert content here...
==October 1, 2014==
 
Today's experiment is a continuation of [http://openwetware.org/wiki/User:Khyra_A._Neal/Notebook/Chem_571/2014/09/30 September 30,2014] lab protocol
===Analysis of Dialysis Solutions===
 
==Bradford Analysis==
* Remove 20 μL of solution from each chamber (10 in all) and run Bradford analysis
** Bradford reagent should be diluted 1:3 with 50mM Tris/50mM NaCl
** Dilute to 1 mL by adding 780 μL  stock 50 mM Tris/NaCl
** Measure UV-Vis from 400 nm - 800 nm using polystyrene cuvettes.
** Run blank of Tris/NaCl buffer
** Run UV-Vis of undialyzed Lysozyme solution with Bradford reagent
 
 
* Transfer remaining dialysis solutions into 20 mL extraction vials.
* Measure Ca<sup>2+</sup> of dialyzed solutions that contain Ca<sup>2+</sup> ion using ISE
** Lysozyme dialyzed in both 50 mM CaCl<sub>2</sub> and 500 μM CaCl2 (a total of 4 solutions)
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Substance'''
| align="center" style="background:#f0f0f0;"|'''Potential (mV)'''
|-
| 50mM CaCl<sub>2</sub> (4) || 80.8
|-
| Lysozyme (4) || 80.3
|-
| 500μM CaCl<sub>2</sub> (3)||28.8
|-
| Lysozyme (3) || 28.1
|-
|}
 
==Fluorescence ==
 
* Dilute each dialyzed lysozyme solution by a factor of 100
* Transfer 100 mL of dialyzed Lysozyme solution to small volume fluorescence cuvette
* Measure fluorescence from 300 nm-550 nm and excitation at 280 nm
* Clean fluorescence cuvette after each use with 1 N HCl, methanol and HPLC water
 
==Dialysis==
 
* Prepare another dialysis chamber using 20,000 g MWCO tubing
* On one side add 1 mL of each:
** HPLC water, 50 mM CaCl<sub>2</sub>, 500 μM CaCl<sub>2</sub>, 0.25 mM HCl, 50 mM NaCl
* On opposite side of well add 1 mL of Lysozyme to the wells directly behind the wells containing HPLC water, 0.25 mM HCl, and 50 mM NaCl
* Add 1 mL of Lysozyme Colloid to the wells directly behind the wells containing 50 mM CaCl<sub>2</sub>, 500 μM CaCl<sub>2</sub>.
* Insert screws  to prevent leaving/evaporation
* Place on low speed shaker for 1 week
 




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October 1, 2014

Today's experiment is a continuation of September 30,2014 lab protocol

Analysis of Dialysis Solutions

Bradford Analysis

  • Remove 20 μL of solution from each chamber (10 in all) and run Bradford analysis
    • Bradford reagent should be diluted 1:3 with 50mM Tris/50mM NaCl
    • Dilute to 1 mL by adding 780 μL stock 50 mM Tris/NaCl
    • Measure UV-Vis from 400 nm - 800 nm using polystyrene cuvettes.
    • Run blank of Tris/NaCl buffer
    • Run UV-Vis of undialyzed Lysozyme solution with Bradford reagent


  • Transfer remaining dialysis solutions into 20 mL extraction vials.
  • Measure Ca2+ of dialyzed solutions that contain Ca2+ ion using ISE
    • Lysozyme dialyzed in both 50 mM CaCl2 and 500 μM CaCl2 (a total of 4 solutions)
Substance Potential (mV)
50mM CaCl2 (4) 80.8
Lysozyme (4) 80.3
500μM CaCl2 (3) 28.8
Lysozyme (3) 28.1

Fluorescence

  • Dilute each dialyzed lysozyme solution by a factor of 100
  • Transfer 100 mL of dialyzed Lysozyme solution to small volume fluorescence cuvette
  • Measure fluorescence from 300 nm-550 nm and excitation at 280 nm
  • Clean fluorescence cuvette after each use with 1 N HCl, methanol and HPLC water

Dialysis

  • Prepare another dialysis chamber using 20,000 g MWCO tubing
  • On one side add 1 mL of each:
    • HPLC water, 50 mM CaCl2, 500 μM CaCl2, 0.25 mM HCl, 50 mM NaCl
  • On opposite side of well add 1 mL of Lysozyme to the wells directly behind the wells containing HPLC water, 0.25 mM HCl, and 50 mM NaCl
  • Add 1 mL of Lysozyme Colloid to the wells directly behind the wells containing 50 mM CaCl2, 500 μM CaCl2.
  • Insert screws to prevent leaving/evaporation
  • Place on low speed shaker for 1 week



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