|
Protein
Purification:
Principles and Practice
|
COURSE
OUTLINE
- 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
-
|