User:Beatriz Gimenez De C./Notebook/571/2014/10/14: Difference between revisions

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* Fluorescence of lysozyme containing solutions
* Fluorescence of lysozyme containing solutions
** Dilute lysozyme sample 100x for each well.  
** Dilute lysozyme sample 100x for each well.  
** Transfer 100 μL to a small volume fluorescence cuvette
** Run fluorescence from 300 nm -550 nm and excitation at 280 nm
** Run fluorescence from 300 nm -550 nm and excitation at 280 nm


* UV-Vis
* UV-Vis
** Run Uv-Vis on protein containing solutions that were dialyzed on [http://openwetware.org/wiki/User:Khyra_A._Neal/Notebook/Chem_571/2014/10/08 October 8, 2014]
** Run Uv-Vis on protein containing solutions obtained from dialisis
** Run from 200 nm -400 nm
** Run from 200 nm -400 nm


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== Prepare 50 mL 66 mM Potassium Phosphate Buffer ==
== Prepare 50 mL 66 mM Potassium Phosphate Buffer ==
* 0.361 g of monobasic potassium phosphate
* Added 0.361 g of monobasic potassium phosphate + 0.105 g of dibasic potassium phosphate to 50 mL volumetric flask + filled with HPLC water
* 0.105 g of dibasic potassium phosphate
* Final pH: 6.319   
* Add to 50 mL volumetric flask and dilute with HPLC water
* Final pH is 6.319   


== Prepare New Dialysis for 30:1 Colloid vs CaCl<sub>2</sub>==
== Prepare New Dialysis for 30:1 Colloid vs CaCl<sub>2</sub>==
* Use 3500 MWCO tubing  
* Use 3500 MWCO tubing  
* Add 1 mL 30:1 colloid solution to 5 cells on one side of chamber
* Add 30:1 colloid solution to 5 cells  
* Add 1 mL 5 μM, 50 μM, 500 μM, 5 mM, and 50 mM CaCl<sub>2</sub> to the opposite side of the chamber (one concentration per cell)
* Add 5 μM, 50 μM, 500 μM, 5 mM, and 50 mM CaCl<sub>2</sub> to the opposite cells
* Secure wells by screwing them to prevent any evaporation
* Follow usual protocol
* Place on low speed shaker overnight


== Prepare Calibration curve for Lysozyme ==
== Prepare Calibration curve for Lysozyme ==

Revision as of 18:53, 31 October 2014

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Tasks

SDS Page

  • Mix 10 μL 0.6 g/L lysozyme with 10μL SDS-PAGE sample buffer in 1.5 mL centrifuge vial
  • Mix 10 μL 0.12 g/L lysozyme with 10μL SDS-PAGE sample buffer in 1.5 mL centrifuge vial
  • Mix 10 μL 30:1 Au/lysozyme colloid with 10μL SDS-PAGE sample buffer in 1.5 mL centrifuge vial
  • Mix 10 μL 0.12 g/L unknown protein with 10 μL SDS-PAGE sample buffer in 1.5 mL centrifuge vial
  • Place in heating block (set at 90 °C) for 5 minutes
  • Store in refrigerator overnight

Analysis of dialyzed from october 8

  • Bradford Analysis
    • Remove 20 μL of each solution + add 200 μL of 1:3 diluted bradford (with 50mM Tris/50mM NaCl)+ further dilute to 1 mL with 50 mM Tris/NaCl
    • Measure UV-Vis from 400 nm - 800 nm (following usual protocol: run blank of Tris/NaCl buffer + undialyzed 0.6 g/L Lysozyme solution with Bradford reagent)
  • Fluorescence of lysozyme containing solutions
    • Dilute lysozyme sample 100x for each well.
    • Run fluorescence from 300 nm -550 nm and excitation at 280 nm
  • UV-Vis
    • Run Uv-Vis on protein containing solutions obtained from dialisis
    • Run from 200 nm -400 nm
  • Ca2+ ISE
    • measured all the samples originally containing 'CaCl2
CaCl2 concentration mV measurement (mV)
5μM 31.8
50μM 40.6
500μM 40.1
5mM 56.6
50mM 78.5

Prepare 50 mL 66 mM Potassium Phosphate Buffer

  • Added 0.361 g of monobasic potassium phosphate + 0.105 g of dibasic potassium phosphate to 50 mL volumetric flask + filled with HPLC water
  • Final pH: 6.319

Prepare New Dialysis for 30:1 Colloid vs CaCl2

  • Use 3500 MWCO tubing
  • Add 30:1 colloid solution to 5 cells
  • Add 5 μM, 50 μM, 500 μM, 5 mM, and 50 mM CaCl2 to the opposite cells
  • Follow usual protocol

Prepare Calibration curve for Lysozyme

  • Bradford analysis for undialysed lysozyme solutions with concentrations 0.12, 0.3, 0.6 and 1 g/L, as described above.


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