User:Alicia Rasines Mazo/Notebook/CHEM-571 Experimental Biological Chemistry/2014/10/07

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Tasks for October 7

  • Run Bradford Analysis of dialysed solutions from Oct. 1
  • Prepare 0.6g/L lysozyme stock
  • Prepare new lysozyme colloid

Bradford Analysis of Dialysed Solutions

Procedure followed as detailed by Dr. Fox on Oct.1

  • 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
    • Recall, 20 μL solution + 200 μL diluted Bradford + 780 μL Tris/NaCl buffer
    • PS cuvettes, measuring 400 - 800 nm
    • Don't forget to run a blank with just Bradford & buffer
    • Don't forget to run your undialyzed Lysozyme stock
  • Transfer remainder of each cell into 20 mL extraction vials

Note: A new 50 mm NaCl 50 mM Tris buffer solution was made by adding:

    • (50×10-3mol/L)×(0.050L)×(121.14g/mol)=0.3030g of Tris buffer
    • (50×10-3mol/L)×(0.050L)×(58.44g/mol)=0.1461g of NaCl
  • And making up to 50 mL with distilled water

pH measurements

Substance pH measurement
0.25 mM HCl (4) 8.062
Lysozyme (4) 8.134

Ca2+ ISE for solutions containing Ca2+

Substance Potential (mV)
Colloid 1 73.5
50 mM CaCl2 (1) 74.9
Colloid 2 51.2
500 μM CaCl2 91.4

Preparation of new lysozyme colloid

A fresh lysozyme stock solution was prepared, given that 1g/L makes the colloid precipitate out of solution. The fresh stock solution is to have a concentration of 0.6g/L, keeping the [Au]:[Lysozyme]=30:1.

  • Transferred 0.5 mL 2.5×10-3M HAuCl4 into 5 culture tubes
  • Max. protein concentration (double volume of protein as gold)=250μM/6=42μM
  • Since final volume of lysozyme stock solution needs to be 25 mL,
    • (42×10-6mol/L)*(0.025L)*(14,307g/mol)=0.015g of lysozyme
    • Made up to 50 mL mark with distilled water
  • Added 1 mL from 42 μM lysozyme stock solution to make [Au:Lysozyme]=30
  • Filled up culture tubes to 5 mL total with water
  • Placed in oven at 80ºC for 4h
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