User:Daniel-Mario Larco/Notebook/AU Photosynthesis Lab/2014/05/29

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Objectives

Today we will focus on two things. First we will continue the protein extraction process form the cells expressed yesterday. Also we will be purifying proteins collected last month

Protein Purification

  1. Disposable filters were used in order to not stain and clog the machine
    1. The filters were open dn the ethanol solution was left to run out
    2. The ethanol was flushed with ~20mL H2O
    3. ~20mL of 100mM phosphate buffer was added once the water ran through
  • this is to avoid buffer crashing out when in contact with ethanol.
    1. Once the buffer ran through, 2.5mL of protein soution was added to the column
    2. Once the flow stopped, 3.5mL of buffer was added and a collection tube was placed under the filter to collect proteins
    3. Once the flow stopped, the collection tube was removed and the waste container was placed under the column
    4. The column was then flushed with ~30mL of buffer
    5. The process was repeated several times

Protein Extraction

  1. The sonicated cells' tube was removed form the freezer and thawed on the bench
  2. Once thawed, the cells were once again sonicated
  3. The solution was placed in the centrifuged and spun at 19000 rpm for 3 hours at 4 degrees Celsius
  4. the supernatant was collected

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
      1. 14.4g Glycine, 3.02g Tris, 1g SDS in 1L H2O
    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. Prepare a 50:50 mixture of BSA and myoglobin in H2O (1mg/mL each). This will serve as a ladder
    2. Load 20uL of ladder and sample in the wells

1. 10uL Sample Buffer + 10uL ladder 2. 10uL Sample Buffer + 1uL ladder + 9uL H2O 4. 10uL Sample Buffer + 10uL Supernatant 6. 10uL Sample Buffer + 1uL Supernatant + 9uL H2O 8. 10uL Sample Buffer + 0.1uL Supernatant + 9.9uL H2O 9. 10uL Sample Buffer + 1uL Supernatant + 9uL H2O 11. 10uL Sample Buffer + 1uL ladder + 9uL H2O 12. 10uL Sample Buffer + 10uL ladder

  1. Perform electrophoresis
    1. Run for 30 minutes at 200V (I need to make sure our power source can do this)
  2. 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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