Lecture 29: Protein Purification: Assays, Initial Steps, Resins
What are the essential features of a useful assay? How does a Western Blot work? Given the results from an initial precipitation experiment propose how you would use it as a purification step. What are the different types of chromatographic resins? How do they work?
Lecture 30: Protein Purification: Ion exchange elutions, dialysis, concentration
How are proteins from ion exchange resins eluted? Explain how to set up a linear and step salt elution. Given experimental data from a gradient elution, explain how you would set up a step elution. Given the experimental conditions from a dialysis or concentration experment be able to determine final protein and/or salt concentration in the sample.
Lecture 31: Protein Purification: Types of Resins, cont. Preparation of Resin, Plumbing
Be able to explain gel filtration, excluded and included volumes and how it relates to purifying a protein with a known molecular mass. Be familiar with the steps involved in preparing and packing resins, and plumbing!
Lecture 32: Protein Purification: Running the Experiment, Resolving Peaks
Provided with an experimental chromatogram explain how you would pool the fractions of interest. Given an experimental protocol which involves a variety of purification steps, explain how you might improve upon the procedure. If you are given a table of a purification scheme which is incomplete, be able to fill in the blanks.
Lecture 33: M13 Phage
Be able to diagram and explain the steps in the M13 phage life cycle. Explain how M13 phage can be turned into a useful cloning vector. Be able to describe the Kunkel method of mutagenesis using M13 cloning vectors
Lecture 34: M13 Phage Display Libraries
Explain how M13 can be used as a phage display library. If provided with a proposed protein or dna random library experiment be able to determine if the experiment is practical (e.g. is it practical to try to construct a random library for a 50 nucleotide stretch of DNA?). What is polyvalent versus monovalent ligand binding? How can monovalent binding be achieved in an M13 library?
Lecture 35: Selective Evolution of Ligands by Exponential Enrichment
Be able to explain how the SELEX system works.
Lecture 36: Protein-Protein Recognition Probed Using a Yeast Transcriptional Activator System
Be able to explain how this system works. What are the characteristics necessary for the host and plasmids? Given a series of constructs and host types, explain what you expect will happen with regard to activation of genes which have a UASGAL
Lecture 37: Molecular Imprinting
Be able to explain the principle behind molecular imprinting.
1998 Dr. Michael Blaber