User:Khyra A. Neal/Notebook/Chem 571/2014/09/23: Difference between revisions

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==Entry title==
===September 23, 2014===
* Insert content here...
 
==SDS-Page and Electrophoresis==
 
The protocol for todays tasks can be found here [http://openwetware.org/wiki/User:Matt_Hartings/Notebook/AU_Biomaterials_Design_Lab/2013/09/25 Electrophoresis]
*Prepare SDS-Page Running Buffer
**10X SDS-Page Running Buffer needs to be diluted by a factor of 10.
**Preparing 2 L →200 mL SDS-Page Running Buffer in 1800 mL of H<sub>2</sub>O
 
*Heat samples prepared on [http://openwetware.org/wiki/User:Khyra_A._Neal/Notebook/Chem_571/2014/09/17 September 17,2014] for 5 minutes at 100°C using the thermocycler.
*Prep Gel and Assemble the Electrophoresis
**Load 15 μL of samples that were prepared on [http://openwetware.org/wiki/User:Khyra_A._Neal/Notebook/Chem_571/2014/09/17 September 17,2014].
*** Well 1: BSA
*** Well 3: Lysozyme
*** Well 5: BSA Colloid
*** Well 7: Lysozyme Colloid
*** Well 9: Soy
*** Well 11: All Blue Protein Standard (provided by Bio-Rad)
 
* Run Electrophoresis for 30 minutes at 200 V
 
* Develop/Stain Gel
**Place gel in Fixative Solution (40% methanol, 10% acetic acid, 50% water) for 30 minutes
**Place gel in Stain Solution (0.025% (w/v) Coomassie Blue, 10% acetic acid, 90% water) for 1 hour
**Place gel in Destain Solution (10% acetic acid, 90% water) for 15 minutes
***Repeat with fresh destain solution twice
 
 
==Analysis of Dialyzed Solutions from [http://openwetware.org/wiki/User:Khyra_A._Neal/Notebook/Chem_571/2014/09/16 September 16,2014]==
'''Bradford Analysis of Soak Solutions Outside of Tubing'''
 
We are conducting a Bradford Assay on the outside soak solutions from the dialysis. The solutions are as follows:
* 3500 g/mol MWCO tubing Soak Solutions
**NaCl
**Glycine
**Pure Soak Water (HPLC Water)
*25k g/mol MWCO tubing in Glycine soak
* In a 1.5 mL centrifuge tube add:
**600 μL of Soak Solution
**200 μL Bradford Reagent (diluted 1:4)
**200 μL Tris/ NaCl Buffer (prepared on [http://openwetware.org/wiki/User:Khyra_A._Neal/Notebook/Chem_571/2014/09/16 September 16,2014] to bring total volume to 1 mL
*Close centrifuge tube, vortex for 5 seconds, and let stand for 5 minutes.  
*Run UV-Vis between 400 nm and 800 nm.  
 
 
 
 
 
 
 




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September 23, 2014

SDS-Page and Electrophoresis

The protocol for todays tasks can be found here Electrophoresis

  • Prepare SDS-Page Running Buffer
    • 10X SDS-Page Running Buffer needs to be diluted by a factor of 10.
    • Preparing 2 L →200 mL SDS-Page Running Buffer in 1800 mL of H2O
  • Heat samples prepared on September 17,2014 for 5 minutes at 100°C using the thermocycler.
  • Prep Gel and Assemble the Electrophoresis
    • Load 15 μL of samples that were prepared on September 17,2014.
      • Well 1: BSA
      • Well 3: Lysozyme
      • Well 5: BSA Colloid
      • Well 7: Lysozyme Colloid
      • Well 9: Soy
      • Well 11: All Blue Protein Standard (provided by Bio-Rad)
  • Run Electrophoresis for 30 minutes at 200 V
  • Develop/Stain Gel
    • Place gel in Fixative Solution (40% methanol, 10% acetic acid, 50% water) for 30 minutes
    • Place gel in Stain Solution (0.025% (w/v) Coomassie Blue, 10% acetic acid, 90% water) for 1 hour
    • Place gel in Destain Solution (10% acetic acid, 90% water) for 15 minutes
      • Repeat with fresh destain solution twice


Analysis of Dialyzed Solutions from September 16,2014

Bradford Analysis of Soak Solutions Outside of Tubing

We are conducting a Bradford Assay on the outside soak solutions from the dialysis. The solutions are as follows:

  • 3500 g/mol MWCO tubing Soak Solutions
    • NaCl
    • Glycine
    • Pure Soak Water (HPLC Water)
  • 25k g/mol MWCO tubing in Glycine soak
  • In a 1.5 mL centrifuge tube add:
    • 600 μL of Soak Solution
    • 200 μL Bradford Reagent (diluted 1:4)
    • 200 μL Tris/ NaCl Buffer (prepared on September 16,2014 to bring total volume to 1 mL
  • Close centrifuge tube, vortex for 5 seconds, and let stand for 5 minutes.
  • Run UV-Vis between 400 nm and 800 nm.






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