User:Alicia Rasines Mazo/Notebook/CHEM-571 Experimental Biological Chemistry/2014/11/04

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Contents

Tasks for November 5

  • To carry out dialysis of 30:1 Colloid solution vs. CaCl2 using MWCO 3500 between 10-50 mM.
  • To prepare solutions for enzyme kinetics

Preparation of solutions for enzyme kinetics

  • The 66 mM potassium phosphate solution prepared on Oct. 14 was used. As recorded, it had pH of 6.319.
  • It was refrigerated overnight
  • Prepared 10 mL 0.01 wt% Micrococcus lysodeikticus Cell Suspension (in freezer) using phosphate buffer. 0.0012 g of Micrococcus lysodeikticus were added.
  • Adjust the cell suspension concentration until the Absorbance at λ = 450 nm is between 0.6 & 0.7. The protocol followed is listed by Sigma-Aldrich and can be reached here: [1]

Preparation of CaCl2 solutions for dialysis

  • A stock solution of 45 mM CaCl2 was prepared by adding 0.2502 g CaCl2 to a 50 mL volumetric flask amd making up to the mark with HPLC water.
  • Serial dilutions were made for 35, 25, 15 and 5 mM CaCl2 mM.

Preparation of 30:1 colloid solution vs CaCl2 using MWCO 3500 dialysis

A new dialysis experiment was prepared using 3,500 MWCO tubing.
The wells were matched up in the following way. Note that 1 mL of the following were added to each well:

  1. 5 mM CaCl2. Opposite to it 30:1 Colloid
  2. 15 mM CaCl2. Opposite to it 30:1 Colloid
  3. 25 mMM CaCl2. Opposite to it 30:1 Colloid
  4. 35 mM CaCl2. Opposite to it 30:1 Colloid
  5. 45 mM CaCl2. Opposite to it 30:1 Colloid
  • Inserted screws to prevent evaporation
  • Placed on low speed shaker for 1 day

Dialysis Data

  • Inspection of the dialysis wells showed that precipitation started to occur at 25 mM of CaCl2, and it was fully precipitated by 35, and 45 mM of CaCl2.

Ca2+ ISE

Substance Potential measurement (mV)
5 mM CaCl2 (1) 56.9
Colloid 1 59.5
15 mM CaCl2 (2) 68.8
Colloid 2 69.6
25 mM CaCl2 (3) 73.7
Colloid 3 72.5
35 mM CaCl2 (4) 76.0
Colloid 4 76.3
45 mM CaCl2 (5) 77.0
Colloid 5 76.4
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