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RECENTLY PUBLISHED:
UNIT 3.11 Free-Flow Electrophoretic Analysis of Endosome Subpopulations in Rat Hepatocytes (Renate Fuchs and Isabella Ellinger, University of Vienna, Vienna, Austria) Endosomes constitute a functionally, morphologically, and biochemically heterogeneous population of intracellular organelles that play a major role in sorting of incoming ligands, receptors, membrane, and lumenal content. This unit provides protocols for labeling and isolation of endosomes of polarized rat hepatocytes. By application of free-flow zone electrophoresis, functional endosomal subcompartments involved in transport to lysosomes are resolved from those involved with transcytosis. Methods to analyze the protein composition or acidification properties of these highly purified endosomes are also described.
UNIT 4.9 Polarization Microscopy (Shinya Inoué, Marine Biological Laboratory, Woods Hole, Mass.). This unit provides an introduction to polarization microscopy, the optics involved, and practical considerations for observing submicroscopic structures. Also included are specific examples for visualization of microtubules in the mitotic spindle, chromatin within maturing spermatids, and the biocrystalline skeletal spicules in larval echinoderms.
UNIT 4.10 Fluorescent Speckle Microscopy (FSM) of Microtubules and Actin in Living Cells (Clare Waterman-Storer, The Scripps Research Institute, La Jolla, Calif.) Fluorescent speckle microscopy (FSM), a combination of conventional wide-field fluorescent light microscopy and digital imaging with a low-noise, charge-coupled devise (CCD) camera, has been developed to allow visualization of assembly/disassembly dynamics, movement, and turnover of macromolecule assemblies in vivo and in vitro. FSM uses a low level of fluorescent subunits to avoid high background. This produces an image of speckled molecules that co-assemble with endogenous molecules, which are followed to characterize dynamic events in living cells.
UNIT 10.8 Matrix Metalloproteinases (L.J. Windsor, University of Indiana School of Dentistry, Indianapolis, Ind., A. Havemose-Poulsen, University of Copenhagen School of Dentistry, Copenhagen, Denmark, S. Yamada, NICDR, NIH, Bethesda, Md., J.G. Lyons, Royal Prince Alfred Hospital, Sydney, Australia, B. Birkedal-Hansen and W. Stetler-Stevenson, NCI, NIH, Bethesda, Md., and H. Birkedal-Hansen, NICDR, NIH, Bethesda, Md.). Matrix metalloproteinases are a class of enzymes that play an important role in the remodeling of the extracellular matrix in development and cancer metastasis. This unit describes a set of methods—cell-mediated dissolution of type I collagen fibrils, direct and reverse zymography, enzyme capture based on a-2 macroglubulin and TIMP-1 and -2, and demonstration of cryptic thiol groups in metalloproteinase precursors—that are used to characterize the functions of matrix metalloproteinases and their inhibitors.
UNIT 11.15 In Vitro Analysis of Peroxisome Protein Import (Stanley
R. Terlecky, Wayne State University School of Medicine, Detroit, Mich.) This
unit describes a quantitative in vitro assay for peroxisomal protein import
that accurately reconstitutes established properties of the pathway. The cell-free
system is ELISA based and employs semi-permeabilized human cells with a biotinylated
peroxisomal targeting signal–containing substrate.
UNIT 11.16 In Vitro Analysis of Chloroplast Protein Import (Lynda Fitzpatrick,
Kenneth Keegstra, Michigan State University, East Lansing, Mich. and Matthew
D. Smith, and Danny J. Schnell, University of Massachusetts, Amherst, Mass.)
This unit describes protocols for isolating chloroplasts from pea (Pisum sativum)
and Arabidopsis thaliana for the study of nuclear-encoded plastid precursor
proteins. Chloroplasts from both preparations are competent for the in vitro
import of recombinant preproteins synthesized using in vitro translation systems
derived from reticulocyte or wheat germ lysates. These assays can be used
to test whether a particular protein is targeted to chloroplasts, for analyzing
the suborganellar location of newly imported preproteins, or to study the
mechanism of import itself.
UNIT 15.5 Analysis of Membrane Traffic in Polarized Epithelial Cells (Joshua H. Lipschutz, Lucy Erin O'Brien, Yoram Altschuler, Dana Avrahami, Yen Nguyen, Kitty Tang, Keith E. Mostov, University of California San Francisco, San Francisco, Calif.). Epithelial cells are characteristically polarized with the apical surface separated from the basolateral surface by tight junctions. Cell surface proteins in these cells are generally expressed at one or the other surface, requiring mechanisms for differential sorting and trafficking. When epithelial cells are grown on a filter with both apical and basolateral surfaces exposed to medium, they polarize so the distributions of cellular and transfected proteins can be studied. This unit includes protocols for pulse-chase labeling of polarized epithelia with and without subsequent biotinylation of newly synthesized proteins, for transfection and isolation of clones expressing the desired protein, for immunofluorescence analysis of the distribution of a protein, and for checking the integrity of a polarized monolayer.
UNIT 15.6 Analysis of Protein Folding and Oxidation in the Endoplasmic Reticulum (Edwin Francis, Robert Daniels, and Daniel N. Hebert, University of Massachusetts, Amherst, Mass.) Proteins that travel through the secretory pathway undergo post-translational folding and oxidation steps that lead to correct conformation of the final protein. This unit focuses on methods for the analysis of folding and oxidation events and the factors responsible for their proper execution. Alkylation and nonreducing SDS-PAGE is use to analyze disulfide bond formation in ER-derived microsomes. If proteins are synthesized under reducing conditions, it is possible to initiate folding by addition of oxidizing agents, thus allowing analysis of factors necessary for the folding process. As folding progresses, the protein of interest shows a change in sensitivity to proteolysis. Coimmunoprecipitation or crosslinking and denaturing immunoprecipitation are used to explore the role of molecular chaperones and other factors. Conformation-specific antibodies can be used to probe folding. In addition, folding can be analyzed in intact or semi-permeabilized adherent or suspension cells.
UNIT 18.4 Quantitative Fluorescence In Situ Hybridization (Q-FISH) (Steven S.S. Poon and Peter M. Lansdorp, Terry Fox Laboratory, Vancouver, B.C., Canada). This unit describes how fluorescently labeled peptide nucleic acid probes can be used to obtain quantitative information about the number of telomere repeat sequences at the ends of individual metaphase chromosomes. Detailed protocols for the preparation of metaphase spreads, Q-FISH procedures, and quantitative digital microscopy set-up and analysis are provided.
FORTHCOMING:
UNIT 6.7 Agarose Gel Electrophoresis of Proteins (Dennis M. Krizek and Margaret E. Rick, Hematology Service, Warren Grant Magnuson Clinical Center, NIH, Bethesda, MD.). This unit describes electrophoretic separation and identification of large proteins from a complex protein mixture. Agarose gel is utilized for the electrophoretic matrix, and detection of proteins is accomplished by transfer of the proteins to a membrane that is probed with specific antibodies and chemiluminescence reagents. Alternatively the protein can be detected in the gel using radiolabeled antibodies and autoradiography.
UNIT 7.10 Protein Labeling by Iodination (Steve Caplan, NICHD-NIH, Bethesda, MD. and Michal Baniyash, Hebrew University-Hadassah Medical School, Jerusalem, Israel). Despite the advances of non-radioactive methods of protein labeling, radioiodination remains an important tool for studying proteins. This unit discusses several commonly used methods for radioiodination of proteins for various purposes. Included are several simple, reliable protocols for radioiodination of cell surface proteins on live cells, radioiodination of purified, solubilized membrane proteins, and radioiodination of purified soluble proteins.
APPENDIX 3B Spectrophotometric Determination of Protein Concentration (Michael H. Simonian, Beckman-Coulter, Fullerton, Calif.). Spectrophotometric methods—absorbance at 280 or 205 nm or fluorescence emission—are easy and useful for determining the concentration of proteins in solution. This unit is an update of the existing unit on this topic.
APPENDIX 3H The Colorimetric Determination and Quantitation
of Total Protein (Randall I. Khron, Pierce Chemical, Rockford, Illinois).
Protein quantification is an important step for handling protein samples for
isolation and characterization; it is a prerequisite step before submitting
proteins for chromatographic, electrophoretic, or immunochemical analysis
and separation. Colorimetric methods are fast and simple, and are not labor-intensive.
This unit describes a number of assays able to detect protein concentrations
in the low microgram to milligram per milliliter ranges in a variety of formats.