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User:Daniel-Mario Larco/Notebook/AU Photosynthesis Lab/2014/07/30

< User:Daniel-Mario Larco‎ | Notebook‎ | AU Photosynthesis Lab‎ | 2014‎ | 07
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Protein Purification

  1. The two sets of fractions were concentrated in the centrifuge and desalted
    1. A total of 80mL of pH 7.2 phosphate buffer was added to each set of fractions prior to centrifuging
    2. Each set was centrifuged until the final volume was ~10mL
  2. The first solution was injected in the SP-column
  3. The elute was collected and stored to be tested
  4. The same was repeated for the second solution

SDS-PAGE

  1. We will run a gel with the solution and past solution to check purity
  2. The gel will be set up in the following order
    1. Myoglobin and BSA ladder
    2. Excess Ruthenium solution
    3. 1st set of fractions from SP (first peak in Q-column 60 minute trial)
    4. Empty
    5. 2nd set of fractions from SP (second peak in Q-column 60 minute trial)
    6. Injection 1 Q-column
    7. Injection 2 " "
    8. Injection 3 " "
    9. Cleaning 1 " "
    10. Cleaning 2 " "
    11. Cleaning 3 " "
    12. Myoglobin and BSA ladder

Gel Electrophoresis

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. Load 18uL of ladder and sample in the wells
  2. Perform electrophoresis
    1. Run for 30 minutes at 200V (I need to make sure our power source can do this)
  3. Develop/Stain your gel
    1. Place gel in Fixative Solution for 30 minutes
    2. Place gel in Stain Solution for 1 hour
    3. Place gel in Destain Solution for 15 minutes
      1. Repeat this step with fresh destain solution 1 more time

Stock Solutions

  1. Protein ladder

-1mg BSA and 1mg Myoglobin in 1mL water -Solution was diluted to 1/10 to be used in gel

  1. 1X Running Buffer

-14.4g Glycine, 1g SDS, 3g Tris in 100mL H2O -Solution was diluted to 1L with water

  1. Fixative Solution

-40 Methanol, 10% Acetic Acid, 50% Water

  1. Stain Solution

-90% water, 10$ Acetic Acid and .0025% Commassie Brilliant Blue

  1. Destain Solution

-90% Water and 10% Acetic Acid