Download presentation
Presentation is loading. Please wait.
Published byIris Hampton Modified over 9 years ago
1
Chapter 8 Tools for protein analysis Progress in science depends on new techniques, new discoveries and new ideas, probably in that order. Sydney Brenner
2
Isolating cells and growing them in culture
3
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
4
A fluorescence-activated cell sorter
5
Microdissection technique for isolating selected cells from tissue slices
6
Cells can be grown in culture (A)Phase-contrast micrograph of fibroblasts in culture. (Live cell imaging video)Live cell imaging video (B)Myoblasts in culture with some cells fusing to form multinucleate muscle cells
7
(C)Oligodendrocyte precursor cells in culture. (D)Tobacco cells from the BY2 cell line grown in liquid culture.
10
Eucaryotic cell lines are a widely used source of homogeneous cells
11
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.
13
Embryonic stem (ES) cells derived from an embryo
14
Reproductive and therapeutic cloning
17
Cells can be fused together to form hybrid cells
18
Hybridoma cell lines provide a permanent source of monoclonal antibodies
20
Purifying proteins
21
The preparative ultracentrifuge Organelles and macromolecules can be separated by ultracentrifugation
22
1000 x g, 10 min 20,000 x g, 20 min 80,000 x g, 1 hr 150,000 x g, 3 hrs Cell fractionation by centrifugation
23
Comparison of velocity sedimentation and equilibrium sedimentation
24
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
25
Proteins can be separated by chromatography Separation of molecules by column chromatography
26
Three types of matrices used for chromatography DEAE-cellulose (positively charged) CM-cellulose and phosphocellulose (negatively charged)
27
Protein purification by chromatography
28
Genetically-engineered tags provide an easy way to purify proteins Epitope tagging for the localization or purification of 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
29
Purification of protein complexes by using a GST-tagged fusion protein
30
Analyzing proteins
31
Proteins can be separated by SDS polyacrylamide-gel electrophoresis
32
The size and subunit composition of a protein can be determined by SDS polyacrylamide-gel electrophoresis
33
Up to 2000 proteins can be resolved on a single gel by two-dimensional polyacrylamide-gel electrophoresis
34
Separation of proteins by isoelectric focusing
35
Two-dimensional polyacrylamide-gel electrophoresis
36
Specific proteins can be detected by Western blotting
37
Selective cleavage of a protein generates a distinctive set of peptide fragments
38
Production of a peptide map, or fingerprint, of a protein
39
Mass spectrometry provides a highly sensitive method for identifying unknown proteins
40
Sets of interacting proteins can be identified by biochemical methods Co-immunoprecipitation Protein affinity chromatography High-density protein arrays
41
Protein-protein interactions can be identified by use of the two-hybrid system
42
Optical methods can monitor protein interactions in real time Fluorescence resonance energy transfer (FRET)
43
Bimolecular fluorescence complementation analysis Schematic representation of the principle of the BiFC assay. Two non- fluorescent 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.
44
The diffraction of X-ray by protein crystals can reveal a protein’s exact structure
45
NMR can be used to determine protein structure in solution
46
Protein sequence and structure provides clues about protein function BLAST search for proteins similar to the human Cdc2 (query) locates maize Cdc2 (subject)
Similar presentations
© 2025 SlidePlayer.com. Inc.
All rights reserved.