Protein Purification for Proteomics: Isolation, Analysis, and Characterization of GFP (Protein Purification)
|Event Date/Time: Mar 12, 2006||End Date/Time: Mar 17, 2006|
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 International)
Filtration Devices including Tangential Flow Ultrafiltration (Millipore, Sartorius, Vivascience,
and introducing the Minimate TFF System, by Pall Life Sciences)
Refrigerated (Sorvall) Centrifuges
Recording UV-Vis Spectrophotometers (Spectronic, Cary)
Filter Fluorometers (Turner Designs, GE Healthcare/Hoefer)
Chromatography: Gel Filtration, Ion-Exchange, & Hydrophobic Interaction (GE Healthcare, BioRad)
Immobilized Metal Ion Affinity Chromatography (Qiagen, BD Biosciences/Clontech)
Fraction Collectors (Gilson, ISCO)
Electrophoresis (SDS, native and isoelectric focusing, 2D) (BioRad, Invitrogen/Novex, GE Healthcare)
Western Blotting (Invitrogen/Novex)
Coomassie Blue and Silver Staining (Sigma-Aldrich)
Ion Exchange HPLC (Applied Biosystems)
Size Exclusion HPLC (Thermo Electron Corporation, Phenomenex)
GE Healthcare Phast Electrophoresis System
Amino Acid Analysis*
Automated Edman Sequencing*
DNA Sequence Analysis*
Mass Spectral Analysis*
*Introduced in problem-solving workshop, Friday
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 followed by a delightful dinner at a local restaurant. 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 a Molecular modeling workshop at the Rutgers Structural Biology Computational Laboratory facilities at Cook College, Rutgers.
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
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
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
The Center for Research and Education in Bioluminescence and Biotechnology (C.R.E.B.B) is a component of Rutgers University, Cook College, and offers a series of continuing education workshops each year featuring nationally renowned presenters. The CREBB mission is to perform basic research on bioluminescence and to utilize bioluminescence (especially the Green Fluorescent Protein) as a tool to educate the scientific and industrial communities in the field of biotechnology.