User:Michaela Harper/Notebook/Biological Chemistry Lab/2011/10/25

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Objective

To run a protein gel to determine how well we have been expressing and purifying the MBP-intein fusion.

Description

Use the Bio Rad Mini Protean system. The instruction manual can be found here. Each group casts its own gel. Make a 12% discontinuous polyacrylamide gel. Each group will need 10 mLs of each gel solution for casting. From there, follow along the directions.

Resolving gel solution for a 12% discontinuous gel (per 10 ml)

  • 3.3mL H2O
  • 4.0mL 30% acrylamide mix
  • 2.5mL 1.5M Tris (pH 8.8)
  • 0.1mL 10% SDS
  • 0.1mL 10% ammonium persulfate
  • 0.004mL TEMED

Stacking gel solution for the gel (per 5mL)

  • 3.4mL H2O
  • 0.83mL 30% acrylamide mix
  • 0.63mL 1.0M Tris (pH 6.8)
  • 0.05mL 10% SDS
  • 0.05mL 10% ammonium persulfate
  • 0.005mL TEMED

This lab will have multiple steps (prepping gels, casting running layer, casting the resolving gel, casting the stacking gel, loading protein samples, running the electrophoresis, staining the gel, rinsing the stained gel).

Group Responsibilities

  • Group 1: Making the resolving gel solution
  • Group 2: Making the stacking gel solution
  • Group 3: Making the staining solution
  • Group 4: 10X running buffer
  • Group 5: 10% Ammonium Persulfate


Group 3 staining gel solution:

  • 0.25 g Coomassie Brilliant Blue R250 in 90 mL MeOH:Water 1:1
  • 10 mL anhydrous acetic acid
  • Filter solution with Whatman No.1 filter


Making the gel

  • Place two glass plates in appropriate clamp with foam to seal the bottom edge.
  • Insert comb to mark bottom edge. Remove comb and fill between the two plates to about 1 cm below bottom edge of comb with resolving gel.
  • Add methanol on top to create an even edge. Let sit 20 minutes.
  • Add stacking gel to the top and insert comb. Let sit 20 minutes.


Making the loading solutions

  • Combine 10 μL ladder and 4 μL loading solution.
  • Combine 16 μL of Peak 1 solution and 4 μL loading solution.
  • Repeat for Peak 2 and 3.


Preparing to run the SDS-PAGE

  • Remove plates from clamp and insert into instrument.
  • Fill all space with buffer solution up to appropriate line for 4 plates.
  • Load ladder and 3 solutions to be run on the gel.


Run the gels, and stain them. Rinse the stain overnight.

Data

Notes


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