User:Daniel-Mario Larco/Notebook/AU Photosynthesis Lab/2014/07/18

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SDS-PAGE

  1. Our protein solution was collected from the cold room
  2. It was filtered using filtration apparatus
  3. It was transferred to a 50mL falcon tube labeled Purified Hb, DML 7/18/2014
  4. We will run a gel with the solution and past solution to check purity
  5. The gel will be set up in the following order
    1. Myoglobin and BSA ladder
    2. Empty
    3. Purified protein solution
    4. 1/10 dilution of Purified protein solution
    5. Empty
    6. Ammonium sulfate treatment pellet dissolved in 50mM Tris
    7. 1/10 dilution of Ammonium sulfate treatment pellet dissolved in 50mM Tris
    8. Empty
    9. Cell pellet dissolved in 50mM Tris
    10. 1/10 dilution of cell pellet dissolved in 50mM Tris
    11. Empty
    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

Results

This gel is oriented so that lane 1 is on the far left. This procedure did not purify the protein as expected At this point, the most successful method has been the combination of Q column and SP column Buffers will be prepared for this method

  1. Prepare buffers
    1. 50mM Tris buffer at pH 8.3
    2. 10mM Sodium Phosphate buffer at pH 7.2
  2. Solutions were filtered using a vacuum filter
  3. Solutions were placed in the autoclave for 1 hour at 121C



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