User:Alicia Rasines Mazo/Notebook/CHEM-571 Experimental Biological Chemistry/2014/10/14: Difference between revisions

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A new dialysis experiment was prepared using '''3,500 MWCO tubing'''. <br.>
A new dialysis experiment was prepared using '''3,500 MWCO tubing'''. <br.>
The wells were matched up in the following way. Note that 1 mL of the following were added to each well:
The wells were matched up in the following way. Note that 1 mL of the following were added to each well:
# 50 mM CaCl<sub>2</sub>. Opposite to it Colloid 1
# 5 μM CaCl<sub>2</sub>. Opposite to it 30:1 Colloid  
# 500 μM CaCl<sub>2</sub>. Opposite to it Colloid 2
# 50 μM CaCl<sub>2</sub>. Opposite to it 30:1 Colloid  
# HPLC water. Opposite to it Lysozyme 3
# 500 μM CaCl<sub>2</sub>. Opposite to it 30:1 Colloid
# 0.25 mM HCl. Opposite to it Lysozyme 4
# 5 mM CaCl<sub>2</sub>. Opposite to it 30:1 Colloid
# 50 mM NaCl. Opposite to it Lysozyme 5
# 50 mM CaCl<sub>2</sub>. Opposite to it 30:1 Colloid
* Inserted screws to prevent evaporation
* Inserted screws to prevent evaporation
* Placed on low speed shaker for 1 week
* Placed on low speed shaker for 1 week
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Revision as of 11:40, 14 October 2014

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Tasks for October 14

  • SDS-PAGE
  • Continuation of dialysis
  • Preparation of 30:1 colloid solution vs CaCl2 using MWCO 3500 dialysis

SDS-PAGE #2

Procedures as listed by Dr. Fox on Oct. 14

  • 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
  • One group prepare additional 1 L of running buffer from 10X concentrate


Note: Ideal concentration for pure protein is 0.5 - 4.0 μg (40 - 60 μg for crude samples).
Using 10 μL of 0.12 g/L will load 1.2 μg protein into the well if entire 20 μL is used.

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

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

  1. 5 μM CaCl2. Opposite to it 30:1 Colloid
  2. 50 μM CaCl2. Opposite to it 30:1 Colloid
  3. 500 μM CaCl2. Opposite to it 30:1 Colloid
  4. 5 mM CaCl2. Opposite to it 30:1 Colloid
  5. 50 mM CaCl2. Opposite to it 30:1 Colloid
  • Inserted screws to prevent evaporation
  • Placed on low speed shaker for 1 week
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