User:Daniel-Mario Larco/Notebook/AU Photosynthesis Lab/2014/07/14

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SDS-PAGE

  1. Our protein solution was collected from the cold room and transferred into a 50mL falcon tube labeled Purified Hb pH7, 7/14/14
  2. We will run a gel with the solution and past solution to check purity
  3. The gel will be set up in the following order
    1. Myoglobin and BSA ladder
    2. Empty
    3. 1/10 dilution of Protein in pH8.3 tris (solution that was injected into Q-Sepharose column)
    4. Elute during injection of Protein in pH8.3 tris into Q-Sepharose column
    5. Elute during cleaning Q-Sepharose column containing Protein in pH8.3 tris with 1M NaCl
    6. Elute from SP-Sepharose column in phosphate buffer pH7.2 during injection (from fractions collected from Q-Sepharose column)
    7. Empty
    8. 1/10 dilution of Ammonium sulfate treatment pellet dissolved in 50mM Tris
    9. 1/10 dilution of purified protein from dialysis over the weekend
    10. 1/100 dilution of purified protein from dialysis over the weekend
    11. 1/10 dilution of centrifuged purified protein from dialysis over the weekend
    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

Lane 1 is furthest left and lane 12 furthest right As we can see, the new method did not provide much purification and the combination of Q- and SP-Sepharose columns remains the best method to date.

Cell Expression preparation

We are making more cells. 1. The correct concentration for dissolving LB Broth Media into water is 25g per liter of water.

- For the 1L LB Broth solution, 25g of LB Broth Media were dissolved into 1L of water with 0.25g of NaCl (because broth mix did not contain any)

- For the 25mL LB Broth solution, 0.625g of LB Broth Media were dissolved into 25mL of water with 0.0625g of NaCl (because broth mix did not contain any)

The solution are placed in the autoclave at 121 degrees C for an hour.

2. In order to prepare the ampicilin solution, 500mg of Ampicilin Sodium Salt were dissolved into 5mL of sterilized water. This solution was prepared in a sterilized eppendorf tube using sterile equipment.

3. Once the solutions cool, 25uL of the ampicilin solution is added to each of the 25ml flasks and 1mL is added to each of the 1L flasks

4. The 25mL flasks were inoculated with K31c Asc Hb (BL21DE3) cells and placed on the shaking incubator at 37C and 225rpm overnight


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