User:Javier Vinals Camallonga/Notebook/Javier Vinals notebook/2013/09/25: Difference between revisions

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==Objective==
==Objective==
Today we'll be determining the molar absorptivities of two different molecules, [http://en.wikipedia.org/wiki/Adenosine adenosine] and [http://en.wikipedia.org/wiki/Inosine inosine]. The data that we generate today will be important when we study [http://en.wikipedia.org/wiki/Adenosine_deaminase adenosine deaminase] (ADA), which converts adenosine to inosine. The difference between these two molecules is that adenosine contains a primary amine whereas inosine contains a carboxy group. Overexpression of this protein causes anemia in humans. A shortage of this protein can lead to severe immuno-defficiency.
To run SDS-PAGE to observe amount of hemoglobin remaining after yesterday's digestion. This should be a complimentary measurement to our UV-Vis analysis from yesterday.
 
 
==Description==
We will be using a Bio-Rad Mini Protean system with pre-cast Mini Protean TGX gels. The manual for this system can be found [http://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_1658100.pdf here] We will be running SDS-PAGE followed by gel development with Coomassie Blue staining.  
 
Below is a short description of how we will proceed. Please refer to the manual for more detailed instructions
 
# Prepare the Gel and Assemble the Electrophoresis Cell
## Remove comb and tape from the gels
## Rinse the wells with running buffer
## Assemble the electrophoresis cell (note diagrams in manual)
## Fill the inner and outer buffer chambers with running buffer
# Prepare and Load Samples
## You prepped your samples yesterday
## Heat your samples for 5 minutes at 100C (in the thermocycler)
## Load 20uL of protein ladder into column 1 of your gel
## Load 20uL of your samples into the appropriate lane of your gel
# Perform electrophoresis
## Run for 30 minutes at 200V (I need to make sure our power source can do this)
# Develop/Stain your 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 this step with fresh destain solution 2 more times


Adenosine and inosine have different absorption spectra. We will be observing changes in UV-Vis spectra to determine changes in concentration of both adenosine and inosine. In order to do this, we will need to know the molar absorptivity (ε) of both of these molecules. Just as each molecule has a characteristic absorption at each wavelength, this (per-wavelength) absorption can be quantified by a molar absorptivity. Or ... for a given concentration a molecule will absorb a very specific amount of light at a precise wavelength. A molecule doesn't have just one molar absorptivity; there is a molar absorptivity to describe each wavelength in a molecular absorbance spectrum.




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Objective

To run SDS-PAGE to observe amount of hemoglobin remaining after yesterday's digestion. This should be a complimentary measurement to our UV-Vis analysis from yesterday.


Description

We will be using a Bio-Rad Mini Protean system with pre-cast Mini Protean TGX gels. The manual for this system can be found here We will be running SDS-PAGE followed by gel development with Coomassie Blue staining.

Below is a short description of how we will proceed. Please refer to the manual for more detailed instructions

  1. Prepare the Gel and Assemble the Electrophoresis Cell
    1. Remove comb and tape from the gels
    2. Rinse the wells with running buffer
    3. Assemble the electrophoresis cell (note diagrams in manual)
    4. Fill the inner and outer buffer chambers with running buffer
  2. Prepare and Load Samples
    1. You prepped your samples yesterday
    2. Heat your samples for 5 minutes at 100C (in the thermocycler)
    3. Load 20uL of protein ladder into column 1 of your gel
    4. Load 20uL of your samples into the appropriate lane of your gel
  3. Perform electrophoresis
    1. Run for 30 minutes at 200V (I need to make sure our power source can do this)
  4. Develop/Stain your gel
    1. Place gel in Fixative Solution (40% methanol, 10% acetic acid, 50% water) for 30 minutes
    2. Place gel in Stain Solution (0.025% (w/v) Coomassie Blue, 10% acetic acid, 90% water) for 1 hour
    3. Place gel in Destain Solution (10% acetic acid, 90% water) for 15 minutes
      1. Repeat this step with fresh destain solution 2 more times



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