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Protein
Purification:
Principles and Practice
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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
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.