Week Five – Protein Gel Electrophoresis

A fool's last words: I wonder if these wires are "hot"...

Monday presentation

The talk will focus on protein structure, sample preparation, gel properties, and principles of discontinuous sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). A presentation with notes is available on line and slides with notes are available as a pdf, formatted for grayscale printing. Make sure that you understand the rationale behind this week's work, and inquire if you need anything clarified.

On your own – before your laboratory session

• Write up the discussion, abstract, and introduction sections for your first research paper, due at the beginning of lab
• Complete prelab #4 before your laboratory session this week
• Peruse the presentation under "Characterizing Erythrocyte Membrane Proteins by SDS-PAGE – Part 2"
• Prepare an outline of work to be done
  • You MUST have all of the volume calculations needed to prepare your samples for electrophoresis
  • Prepare to apply any or all of the skills and concepts covered in previous laboratory sessions
  • Be able to describe the overall objectives and specific objectives of this week's work
  • Be prepared to describe the structural organization of proteins and the difference between a protein and a polypeptide
  • Be prepared to describe the basis for separation of macromolecules by SDS-PAGE
  • Be able to explain why your protein samples must be denatured, and the purpose of SDS and other sample buffer components
  • Be prepared to describe molecular mass and define what is meant by a Dalton
  • Be able to describe the purpose of the molecular mass standards and how they are used
  • Be prepared to describe what is meant by %T and %C in reference to polyacrylamide gels
  • Be able to describe the difference between a separating gel and a stacking gel
  • Be prepared to explain why the same protein samples should be run on gels of different density (%T)

Pre-laboratory orientation

• Safety concerns, including hazards of acrylamide
• Organization of teams
• Comments on division of labor and record keeping
• Suggestions for sample preparation
• Instructions for finishing gels and mounting a gel in a stand
• Recommendations for loading and running gels

Laboratory work this week

You will complete the preparation of acrylamide gels for SDS-PAGE by preparing and pouring the stacking solution. You will prepare your protein samples (aliquots from the previous laboratory session) for electrophoresis. You will set up, load, and run your gel(s), take them down, and start the staining process.

Follow-up work

• Download and print off your gel photos when they become available; you need 2 copies of each gel photo per person, and need not print in color
• Write up the materials and methods to the second study, to be submitted in the laboratory next week
• Start on your preparation for the sixth week of the course (Week 6 schedule page)
• Prelab #5 is to be completed and submitted electronically by midnight before your lab day next week