Protein Purification
- The two sets of fractions were concentrated in the centrifuge and desalted
- A total of 80mL of pH 7.2 phosphate buffer was added to each set of fractions prior to centrifuging
- Each set was centrifuged until the final volume was ~10mL
- The first solution was injected in the SP-column
- The elute was collected and stored to be tested
- The same was repeated for the second solution
SDS-PAGE
- We will run a gel with the solution and past solution to check purity
- The gel will be set up in the following order
- Myoglobin and BSA ladder
- Excess Ruthenium solution
- 1st set of fractions from SP (first peak in Q-column 60 minute trial)
- Empty
- 2nd set of fractions from SP (second peak in Q-column 60 minute trial)
- Injection 1 Q-column
- Injection 2 " "
- Injection 3 " "
- Cleaning 1 " "
- Cleaning 2 " "
- Cleaning 3 " "
- 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
- Prepare the Gel and Assemble the Electrophoresis Cell
- Remove comb and tape from the gels
- Rinse the wells with running buffer
- Load 18uL of ladder and sample in the wells
- Perform electrophoresis
- Run for 30 minutes at 200V (I need to make sure our power source can do this)
- Develop/Stain your gel
- Place gel in Fixative Solution for 30 minutes
- Place gel in Stain Solution for 1 hour
- Place gel in Destain Solution for 15 minutes
- Repeat this step with fresh destain solution 1 more time
Stock Solutions
- Protein ladder
-1mg BSA and 1mg Myoglobin in 1mL water
-Solution was diluted to 1/10 to be used in gel
- 1X Running Buffer
-14.4g Glycine, 1g SDS, 3g Tris in 100mL H2O
-Solution was diluted to 1L with water
- Fixative Solution
-40 Methanol, 10% Acetic Acid, 50% Water
- Stain Solution
-90% water, 10$ Acetic Acid and .0025% Commassie Brilliant Blue
- Destain Solution
-90% Water and 10% Acetic Acid
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