User:Floriane Briere/Notebook/CHEM-496/2011/10/25

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Objective

Today's experiment consists on performing a gel electrophoresis on MPB-intein fusion which have been expressed and purified by groups 2 and 3. This experiment will help us to determine whether or not the expressions and purifications worked well.

To do so, we are going to run a SDS-PAGE using a 12% discontinuous polyacrylamide gel.

  1. The SDS-PAGE technique permits to separate proteins according to their molecular weight. The SDS (Sodium Dodecyl Sulfate) is an anionique surfactant which is going to denature the proteins and add negative charges on it; so proteins are going to migrate through the gel according to their molecular weight/negative charge ratio.
  2. The discontinuous polyacrylamide gel is composed of two parts:
    1. The stacking gel: compare to the resolving gel, the stacking gel has a lower concentration of acrymalide and a lower pH. It allows to concentrate the proteins into a thight band.
    2. The resolving gel: it permits to separate the proteins which are going to migrate through the gel according to their molecular weight.

The protocol was esthablished using this manuel.

Protocol

  1. Prepare the stock solutions:
    1. 30% acrylamide mix:
    2. 10% SDS
    3. 10% APS (1ml): mix 100mg of APS with 1ml of water
    4. Resolving gel solution (10ml): 3.3ml of H2O + 4.0ml of 30% acrylamide mix + 2.5ml of Tris buffer (1.5M; pH 8.8) + 0.1ml 10% SDS
    5. Stacking gel solution (5ml): 3.4ml H2O + 0.83mL 30% acrylamide mix + 0.63mL of Tris buffer (1.0M; pH 6.8) + 0.05mL 10% SDS
  2. Pour the discontinuous gel:
    1. Add to 10ml of the resolving gel solution 0.1ml APS + 0.004ml TEMED
    2. Mark the glass plate 1 cm below the comb teeth
    3. Pour with the complete resolving gel solution to the mark
    4. Complete with ethanol (or water) so to flatten the top of the resolving gel
    5. Let the gel polymerize for 20 minutes
    6. Remove the ethanol
    7. Add to 5ml of the stacking gel solution 0.05ml of APS + 0.005ml of TEMED
    8. Pour with the complete stacking gel solution until the short plate is reached
    9. Add the comb
    10. Let the gel polymerize for 20 minutes
  3. Prepare the samples:
    1. Tube 1: 10µl of ladder (blue) + 4µl of loading buffer (orange)
    2. Tube 2: 16µl of protein 1 + 4µl of loading buffer (orange)
    3. Tube 3: 16µl of protein 2 + 4µl of loading buffer (orange)
    4. Tube 4: 16µl of protein 3 + 4µl of loading buffer (orange)
  4. Run the SDS-PAGE electrophoresis:
    1. Remove the comb
    2. Load the sample into the wells
    3. Run the gel for 35 minutes at 200 volts
  5. Stain the polyacrylamide gel:
    1. Cover the gel with stain (blue)
    2. Leave overnight on the shaker



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