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Protein Purification: Principles and Practice


2010 Course Dates
January 3-8, 2010
March 14-19, 2010

A 5 ½ day hands-on laboratory course in which participants will learn skills to effectively design separation and purification strategies for downstream processing. This course uses the remarkable Green-Fluorescent Protein (GFP), a novel marker for gene expression, as the source material. While this is a general course applicable to all proteins, we use GFP as a highly effective visual teaching tool.

More than 1200 scientists from around the world have strongly recommended this intensive course as an opportunity to develop protein research and analytical skills in a retreat setting. Participants work hard, identify and solve problems in the lab and enjoy comaraderie and good food and drink with colleagues.

This five and one half day laboratory course covers a wide variety of conventional methods for protein isolation, purification, and characterization. The course format integrates hands-on laboratory exercises with classroom lectures, demonstrations, study breaks, and short take-home assignments.

A special feature of the course is that all laboratory work will be performed on the same starting sample (Aequorea GFP or recombinant GFP), which will be purified from an exceedingly crude form (starting with tissue or bacterial cell extraction) to near homogeneity as judged by high performance liquid chromatography (HPLC), SDS gel electrophoresis, isoelectric focusing, and western blotting. This feature provides a continuity of purpose, integrating dozens of preparative and analytical protein techniques in a way that few competing courses can match.

A problem-solving approach will be used throughout the course. Under the guidance of experienced lab instructors, participants will work in groups of three to plan their own protocols, analyze data, and interpret results. A student-teacher ratio not greater than 8:1 will be maintained and the faculty coordinators will be present throughout the course.


SUNDAY 3:00 p.m. - 9:00 p.m.

- Registration and Reception 3:00 p.m.-4:00 p.m.
- Course Introduction and Overview 4:00 p.m.-7:00 pm.
- Dinner at a nice local restaurant: 7:00 p.m. - 9:00 p.m.

MONDAY 7:30 a.m. - 8:00 p.m.

- Lecture: Protein Structure
- Laboratory Introduction and Overview
- Morning Laboratory Exercises: Filtration, Precipitation with Ammonium Sulfate, Centrifugation, Fluorimetric and Spectrophotometric Assays, and Gel Filtration Chromatography
- Lecture: General Preparative Methods
- Evening Laboratory Exercises: Do Biochemical Assays, Plot Gel Filtration Data, Select Ion Exchange Conditions, Dialysis and Begin Purification Table

TUESDAY 7:30 a.m. - 5:30 p.m.

- Lecture: Open Column Purification
- Demonstration: Analytical Gel Filtration
- Morning Laboratory Exercises: Centrifuge Dialyzed Sample, Begin Ion-Exchange Chromatography
- Afternoon Laboratory Exercises: Choose HIC Matrix and Eluting Solvent, Assay Ion-Exchange Fractions, Plot Data

WEDNESDAY 7:30 a.m. - 8:30 p.m.

- Morning Laboratory Exercises: Run HIC on Peak Ion-Exchange Fractions
- Lecture: HPLC Theory
- Morning Laboratory Exercises: Assay HIC Fractions
- Afternoon Laboratory Exercises: Concentrate and Desalt Samples, Purify by SEC-HPLC and Calculate Molecular Weight, Analyze Pure GFP Spectrally
- Lecture: Electrophoresis Evening Laboratory Exercises:
- Demonstration: Tangential Flow Ultrafiltration (Millipore, Sartorius, and the Pall Minimate TFF System)
- Demonstration: Three Phase Purification of Proteins using t-Butanol

Interactive Workshops
1. Affinity Chromatography of His-tagged Protein on Qiagen Nickel Column
2. Western Blotting on the Invitrogen/Novex System
3. Titration Curve on GE Healthcare "Phast" System

THURSDAY 7:30 a.m. - 5:30 p.m.

- Demonstration: Preparative PAGE on BioRad Prep Cell, 2 Dimentional Electrophoresis on Zoom IPGRunner System
- Morning Laboratory Exercises: Pour SDS Running Gel. Load Isoelectric Focusing Gel
- Demonstration: "Phast" System, Native Gel with Western Blot
- Lecture: Analysis of Protein Structure
- Laboratory Exercises (continued): Pour SDS Stacking Gel, Stain IEF Gel
- Afternoon Laboratory Exercises: Run SDS Gel, Stain and Destain SDS Gels

FRIDAY 7:30 a.m. - 4:00 p.m.

- Lecture: Molecular Biology of GFP or HPLC of Proteins and Peptides
- Morning Laboratory Exercises: Analyze SDS Gels, Plot SDS Molecular Weight Data, Analyze IEF Gel, Determine Isoelectric Point, Analyze "Phast" IEF, Complete Purification Table
- Interactive Workshop: Determination of Chromopeptide Structure:   Analyze Chromopeptide Purification Data.
  Discuss and Analyze:
(1) Amino Acid Composition Data,
(2) Edman Sequencing Data,
(3) Pronase/Carboxypeptidase Data
(4) DNA Sequence Data and
(5) Model Compound Analysis.
-Molecular modeling workshop: Rutgers Structural Biology Computational Laboratory

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