Protein Purification: Isolation, Analysis, and Characterization of GFP (Protein Purification)

Venue: Cook College, Rutgers University

Location: New Brunswick, New Jersey,

Event Date/Time: Jan 13, 2002 End Date/Time: Jan 18, 2002
Early Registration Date: Dec 14, 2001
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The State University of New Jersey
Cook Campus at New Brunswick
The Center for Research and Education in Bioluminescence and Biotechnology
Protein Purification: Isolation, Analysis, and Characterization of GFP. A Five and One-Half Day Hands-On Laboratory Course using the remarkable Green-Fluorescent Protein (GFP), A Novel Marker For Gene Expression, as the source material.

January 13-18, 2002
March 17-22, 2002
and Summer, 2002 (TBA)

More than 600 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 comraderie 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.

Techniques and Instruments You Will Use

* Tissue Homogenizers (Omni)
* Filtration Devices including Tangential Flow Ultrafiltration (Millipore, Sartorius)
* Refrigerated (Sorvall) Centrifuges
* Recording UV-Vis Spectrophotometers (Beckman)
* Filter Fluorometers (Turner Designs, Hoefer)
* Chromatography: Gel Filtration, Ion-Exchange, & Hydrophobic Interaction (Pharmacia, BioRad)
* Fraction Collectors (Gilson, ISCO)
* Column Monitors
* Electrophoresis (SDS, native and isoelectric focusing) (BioRad, Novex, Pharmacia)
* Western Blotting (Novex)
* Coomassie Blue and Silver Staining
* Ion Exchange HPLC (PE Biosystems)
* Size Exclusion HPLC (ThermoQuest, Phenomenex)
* Pharmacia Phast Electrophoresis System
* Amino Acid Analysis*
* Automated Edman Sequencing*
* DNA Sequence Analysis*
* Peptide Mapping*
* Mass Spectral Analysis*

*Introduced in problem-solving workshop, Friday

Course Format

Course participants will extract an easily visualized chromoprotein, the green-fluorescent protein, (Science vol. 263 pp. 802-805,1994) from a frozen tissue sample or bacterial cell pellets, clarify the extract, and then concentrate and purify the protein by "salting out". Gel filtration, ion exchange, hydrophobic interaction, and size exclusion HPLC chromatography will then be employed to extensively purify the desired protein (GFP) from the crude extract. The unique nature of this brilliantly fluorescent protein allows you to follow all phases of the purification with a simple hand-held mineral light, enhancing the students' understanding of each process. The purified protein will be characterized by SDS and native gel electrophoresis, isoelectric focusing, ion exchange HPLC, size exclusion HPLC, and Western blotting. Each group will prepare a detailed purification table and graphs (homework assignments), and will characterize the protein with respect to purity, charge, molecular weight, isoelectric point, unique spectral features, subunit composition, isoprotein composition, and the chemical nature of the chromogenic peptide. This course integrates lecture and laboratory sessions to provide a comprehensive learning experience. The course begins with an introductory lecture on Sunday afternoon. Everyone is strongly encouraged to attend this session, but participants who cannot arrive for the Sunday lecture may begin the course on Monday morning (at the laboratory location). The course concludes Friday afternoon with an interactive problem-solving workshop and tour of the mass spectroscopy facilities at Cook College, Rutgers.


Additional Information

Course Outline SUNDAY 3:00 p.m. - 7:00 p.m. - Registration and Reception 3:00 p.m.-4:00 p.m. - Course Introduction and Overview 4:00 p.m.-6:00 pm. - Dinner: 6:00 p.m. - 7:00 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, 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 BioCad Ion Exchange - 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: Ion Exchange Chromatography on Pharmacia "FPLC" system - Demonstration: Tangential Flow Ultrafiltration - 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 Novex System 3. Titration Curve on Pharmacia "Phast" System THURSDAY 7:30 a.m. - 5:30 p.m. - Demonstration: Preparative PAGE on BioRad Prep Cell - Morning Laboratory Exercises: Pour SDS Running Gel. Load Isoelectric Focusing Gel - Demonstration: "Phast" System, Native Gel with Western Blot - Lecture: HPLC of Proteins and Peptides - 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 - Morning Laboratory Exercises:Analyze SDS Gels, Plot SDS Molecular Weight Data, Analyze IEF Gel, Determine Isoelectric Point, Analyze "Phast" IEF, Complete Purification Table - Lecture: Analysis of Protein Structure - 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. - Tour: Rutgers University Mass Spectroscopy Facility