User:Daniel-Mario Larco/Notebook/AU Photosynthesis Lab/2014/07/23

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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. Initial protein solution
    3. Elute during injection in Q-column 7/22
    4. Elute during cleaning Q-column w/ 1M NaCl 7/22
    5. Elute from SP-column during injection 1 7/22
    6. Elute from SP-column during injection 2 7/22
    7. Elute during cleaning SP-column w/ 1M NaCl 7/22
    8. Elute before and after collection of fractions (frac 1-3, 11-14, 22, 23) 7/22
    9. Fractions 11-13, 0-160mM NaCl from Q-column 7/21
    10. Elute from fractions 11-13 from SP-column 7/21
    11. Fractions 14-23, 160-300mM NaCl from Q-column 7/21
    12. Elute from fractions 14-23 from SP-column 7/21

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

The corner with the nick is the top corner above lane 1. The gel showed that the elutes from the SP-column were purified from the original solution but not as pure as desired. The elutes were combined and concentrated in a centrifugal device at 3680rpm until the final volume was ~10mL



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