User:Matt Hartings/Notebook/AU Biomaterials Design Lab/2011/10/25

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Objective

Today we are going to be running a protein gel to determine how well we have been expressing and purifying the MBP-intein fusion. (Groups 2 and 3 have thus far done the expression and purification).

We will be using the Bio Rad Mini Protean system. The instruction manual can be found here. Each group will have to cast its own gel. We will be making a discontinuous polyacrylamide gel (so follow those instructions). We will be making a 12% 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 we'll run (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
    • NOTE: These are just the volumes of solutions you need to make these. Please follow the protocol in the instruction manual for how to put each of these solutions together to make the final casting gel solution!!!!

Because we need to make lots of solutions, each group will be responsible for putting together 1 or more solutions. Please pay attention to what you need to make and begin making these buffers and solutions IMMEDIATELY upon entering lab. Everyone will be relying on you for quick and efficient preparation of buffers. 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). Please be efficient with your preparation! The best way to do this is to be prepared coming into class.

Group Responsibilities

  • Group 1: Making the resolving gel solution
  • Group 2: Making the stacking gel solution
  • Group 3: 50mL of 30% acrylamide mix (see instruction manual)
  • Group 4: 10X running buffer
  • Group 5: 10% Ammonium Persulfate

Again, be quick. Be courteous.

Once we have cast our gels. Tamra will go over some proper methods for loading and running the SDS-PAGE. After we run the gels, we will stain them and rinse the stain overnight.

Description

  1. Add experimental record here. Include what, how, and why...

Data

  • Add data and results here...

Notes

This area is for any observations or conclusions that you would like to note.


Use categories like tags. Change the "Course" category to the one corresponding to your course. The "Miscellaneous" tag can be used for particular experiments, as instructed by your professor. Please be sure to change or delete this tag as required so that the categories remain well organized.




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