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Chapter 8
Tools for protein analysis
Progress in science depends on new techniques, new discoveries and new ideas,
probably in that order.
Sydney Brenner
Isolating cells and growing them in culture
Cells can be isolated from a tissue and separated
into different types
Animal tissues – treatment with proteolytic enzymes (such as trypsin
and collagenase) and chelating agents (such as EDTA)
Plant tissues – treatment with pectinase
A fluorescence-activated cell sorter
Microdissection technique for isolating selected cells from tissue slices
Cells can be grown in culture
(A)
Phase-contrast micrograph of fibroblasts in culture. (Live cell imaging video)
(B)
Myoblasts in culture with some cells fusing to form multinucleate muscle cells
(C)
Oligodendrocyte precursor cells in culture.
(D)
Tobacco cells from the BY2 cell line grown in liquid culture.
Eucaryotic cell lines are a widely used source of homogeneous cells
Her name was Henrietta Lacks, but scientists know her as HeLa. She was a poor Southern tobacco
farmer who worked the same land as her slave ancestors, yet her cells—taken without her knowledge—
became one of the most important tools in medicine. The first “immortal” human cells grown in culture,
they are still alive today, though she has been dead for more than sixty years. If you could pile all HeLa
cells ever grown onto a scale, they’d weigh more than 50 million metric tons—as much as a hundred
Empire State Buildings. HeLa cells were vital for developing the polio vaccine; uncovered secrets of
cancer, viruses, and the effects of the atom bomb; helped lead to important advances like in vitro
fertilization, cloning, and gene mapping; and have been bought and sold by the billions.
Embryonic stem (ES) cells derived from an embryo
Reproductive and therapeutic cloning
Cells can be fused together to form hybrid cells
Hybridoma cell lines provide a permanent source of monoclonal antibodies
Purifying proteins
Organelles and macromolecules can be separated by ultracentrifugation
The preparative ultracentrifuge
Cell fractionation
by centrifugation
1000 x g, 10 min
20,000 x g, 20 min
80,000 x g, 1 hr
150,000 x g, 3 hrs
Comparison of velocity sedimentation and equilibrium sedimentation
Cell extracts provide accessible systems to study cell functions
Functions of mitochondria, chloroplasts, and vesicles formed from fragments
of the rough and smooth endoplasmic reticulum (microsomes)
Extracts of Xenopus laevis oocytes used in understanding of processes such as
the cell-division cycle, separation of chromosomes, and vesicular transport
Proteins can be separated by chromatography
Separation of molecules by column chromatography
DEAE-cellulose (positively charged)
CM-cellulose and phosphocellulose (negatively charged)
Three types of matrices used for chromatography
Protein purification by chromatography
Genetically-engineered tags provide an easy way to purify proteins
- antigenic determinant, or epitope, recognized
by a specific antibody
- short string of histidines (His-tag) binds to an
affinity column containing immobilized nickel
ions
- Glutathione S-transferase (GST) fusion protein
binds to an affinity column containing
glutathione
Epitope tagging for the localization or purification of proteins
Purification of protein complexes by using a GST-tagged fusion protein
Analyzing proteins
Proteins can be separated by SDS polyacrylamide-gel electrophoresis
The size and subunit composition of a protein can be determined by
SDS polyacrylamide-gel electrophoresis
Up to 2000 proteins can be resolved on a single gel by
two-dimensional polyacrylamide-gel electrophoresis
Separation of proteins by isoelectric focusing
Two-dimensional polyacrylamide-gel electrophoresis
Specific proteins can be detected by Western blotting
Selective cleavage of a protein generates a distinctive set of peptide fragments
Production of a peptide map, or fingerprint, of a protein
Mass spectrometry provides a highly sensitive method for identifying unknown proteins
Sets of interacting proteins can be identified by biochemical methods
Co-immunoprecipitation
Protein affinity chromatography
High-density protein arrays
Protein-protein interactions can be identified by use of the two-hybrid system
Optical methods can monitor protein interactions in real time
Fluorescence resonance energy transfer (FRET)
Bimolecular fluorescence complementation analysis
Schematic representation of the principle of the BiFC assay. Two nonfluorescent fragments (YN and YC) of the yellow fluorescent protein (YFP)
are fused to putative interaction partners (A and B).The association of the
interaction partners allows formation of a bimolecular fluorescent complex.
The image shows an example of a complex formed by nuclear proteins.
The diffraction of X-ray by protein crystals can reveal a protein’s exact structure
NMR can be used to determine protein structure in solution
Protein sequence and structure provides clues about protein function
BLAST search for proteins similar to the human Cdc2 (query) locates maize Cdc2 (subject)