User:Alicia Rasines Mazo/Notebook/CHEM-571 Experimental Biological Chemistry/2014/10/28: Difference between revisions

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===0.6 g/L lysozyme vs CaCl<sub>2</sub> using 3500 MWCO dialysis data===
===0.6 g/L lysozyme vs CaCl<sub>2</sub> using 3500 MWCO dialysis data===
<u>Bradford Analysis</u>
*Only running bradford analysis of protein-containing solutions, that is Lys 1, 2, 3, 4 and 5, since the protein cannot diffuse through 3500 MWCO.
* Remove 50 μL of solution from each chamber (7 in all) and run Bradford analysis
**Bradford reagent should be diluted 1:3 with 50mM Tris/50mM NaCl
**Recall, 50 μL solution + 200 μL diluted Bradford + 750 μL Tris/NaCl buffer
**PS cuvettes, measuring 400 - 800 nm
<u>Ca2+ ISE</u>
<u>Ca2+ ISE</u>
{| {{table}}
{| {{table}}
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| align="center" style="background:#f0f0f0;"|'''mV measurement (mV)'''
| align="center" style="background:#f0f0f0;"|'''mV measurement (mV)'''
|-
|-
| 5μM (1) || 12.9
| 5μM (1) || 11.8
|-
|-
| Lys (1) ||  
| Lys (1) || 4.6
|-
|-
| 50μM (2) ||
| 50μM (2) || 4.2
|-
|-
| Lys (2) ||  
| Lys (2) || 0.1
|-
|-
| 500μM (3) ||  
| 500μM (3) || 27.4
|-
|-
| Lys (3) ||  
| Lys (3) || 24.4
|-
|-
| 5mM (4) ||  
| 5mM (4) || 52.1
|-
|-
| Lys (4) ||  
| Lys (4) || 51.3
|-
|-
| 50mM (5)||  
| 50mM (5)|| 77.3
|-
|-
| Lys (5) ||  
| Lys (5) || 77.0
|-
|-
|}
|}
<u>UV-Vis Absorption</u>
<u>UV-Vis Absorption</u> <br.>
Transfer 100 μL of 100:1 diluted dialysed lysozume to a small volume UV cuvette & measure UV absorption
**Be sure your cuvette is clean before hand.  Use SDS, HCl, HPLC, & methanol washes
**Measured entire 200 - 800 nm range. (Care about peak at 280 nm. Need to compare to fluorescence data to see how the binding is affected)
<u>Fluorescence</u>
<u>Fluorescence</u>
*Measured fluorescence of dyalised solutions
*Fluorescence (glass microcuvette & 100 fold diluted) for all protein solutions
**10μL of lysozyme were diluted to 1mL by adding HPLC. (Remember to account for dilution factor later on)
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Revision as of 12:10, 29 October 2014

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Tasks for October 28

  • To continue analysis of dialysed 0.6 g/ L Lysozyme vs KI using 3500 MWCO
  • To check I- ISE values obtained on OCt. 21 and measure KI content of dialysed solutions
  • Set up dialysis of 0.6 g/L lysozyme vs CaCl2 using 3500 MWCO

I- ISE

KI concentration mV measurement (mV) (21 Oct.) mV measurement (mV) (28 Oct.)
2mM -198.3 -192.8
5 mM -217.3 -206.8
10 mM -237.0 -224.2
25 mM -247.4 -253.0
50mM -278.0 -276.2

0.6 g/L lysozyme vs CaCl2 using 3500 MWCO dialysis data

Bradford Analysis

  • Only running bradford analysis of protein-containing solutions, that is Lys 1, 2, 3, 4 and 5, since the protein cannot diffuse through 3500 MWCO.
  • Remove 50 μL of solution from each chamber (7 in all) and run Bradford analysis
    • Bradford reagent should be diluted 1:3 with 50mM Tris/50mM NaCl
    • Recall, 50 μL solution + 200 μL diluted Bradford + 750 μL Tris/NaCl buffer
    • PS cuvettes, measuring 400 - 800 nm

Ca2+ ISE

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

UV-Vis Absorption UV-Vis Absorption <br.> Transfer 100 μL of 100:1 diluted dialysed lysozume to a small volume UV cuvette & measure UV absorption

    • Be sure your cuvette is clean before hand. Use SDS, HCl, HPLC, & methanol washes
    • Measured entire 200 - 800 nm range. (Care about peak at 280 nm. Need to compare to fluorescence data to see how the binding is affected)

Fluorescence Fluorescence

  • Measured fluorescence of dyalised solutions
  • Fluorescence (glass microcuvette & 100 fold diluted) for all protein solutions
    • 10μL of lysozyme were diluted to 1mL by adding HPLC. (Remember to account for dilution factor later on)
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