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)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

The preparative ultracentrifuge Organelles and macromolecules can be separated by ultracentrifugation

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

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

Three types of matrices used for chromatography DEAE-cellulose (positively charged) CM-cellulose and phosphocellulose (negatively charged)

Protein purification by chromatography

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

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 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.

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)