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Chapter 57 Nonradioactive Methods for Labeling and Identifying Membrane Surface Proteins
CHAPTER 57
Nonradioactive Methods for Labeling and Identifying Membrane Surface Proteins William L. Dentler Department of Physiology and Cell Biology University of Kansas Lawrence, Kansas 66045
I. Introduction 11. Methods
A. Labeling Tetrahymena Cilia with NHS-LC Biotin B. Alkaline Phosphatase-Streptavidin Staining of Biotinylated Protein C . Labeling Blotted Proteins with Concanavalin A References
I. Introduction In addition to motility, cilia provide surfaces on which a variety of cell surface molecules are displayed for cell-cell interactions during mating, chemoreception, and a variety of cellular functions. For a typical biflagellate Chlamydornonus cell, the surface area of the flagellar membrane is approximately 15 pm2, about 0.3% of the total surface area of the cell. For Tetrahymena pyriformis, which contains approximately 600 cilia per cell, the total surface area of the membrane can be greater than 40% of the total cell surface area. At present, we have little understanding of the function and identity of most of the ciliary surface proteins, other than the fact that many are present. In a study of Tetrahymena thermophila ciliary membrane proteins, we identified more than 40 ciliary membrane surface proteins, at least 16 of which also bind concanavalin A (Dentler, 1992). Other than 36- and 50-kDa polypeptides that appear to be associated with a microtubule-membrane bridge, the functions of these proteins remain unknown. METHODS IN CELL BIOLOGY. VOL. 47
Copyright 0 1995 by Academic Press. Inc. All rights of reproduction m any form reserved
407
408
William L. Dentler
Putative membrane surface proteins can be identified by iodination (Bloodgood and Workman, 1984; Williams et al., 1980) followed by resolution of proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography. There is, however, concern about this method due to its potential to label cytoplasmic proteins (Thompson et al., 1987) and the use of 1251, In contrast to iodination, labeling with biotin provides a gentle and nonradioactive method to label surface proteins in a variety of cells (see Goodloe-Holland and Luna, 1987). Biotinylation involves the covalent coupling of biotin to a protein or other molecule and the subsequent binding of biotin to avidin or the more specific streptavidin, with which biotin forms an extremely high affinity bond (with a dissociation constant of -lo-'' M). Avidin or streptavidin can be coupled to alkaline phosphatase or other reagents that produce a colorimetric reaction used to identify biotinylated proteins bound to nitrocellulose. Although streptavidin-gold particles should be useful in identifying biotinylated proteins by electron microscopy, we have had relatively little success in obtaining convincing data using this procedure. A variety of compounds, some of which are reversible, can be used to link biotin to proteins, carbohydrates, and nucleic acids. One of the best sources for these compounds is Pierce Chemical, and the reader is encouraged to consult their catalog for new compounds and labeling procedures. We use sulfosuccinimidyl 6-(biotinamido) hexanoate (NHS-LC biotin), which reacts with primary amines (generally lysine epsilon groups) to form a stable covalent amide bond. Reactions are favored in slightly alkaline media, in which the primary amines are unprotonated. The NHS-LC biotin is water soluble and does not penetrate the ciliary membrane to label axonemal or cytoplasmic proteins (Dentler, 1992). The sensitivity of the reagent depends on the detection methods used after SDS-PAGE and blotting to nitrocellulose. Adequate labeling, comparable to iodination, can be achieved using horseradish peroxidase (HRP)-streptavidin to stain the biotin, but more than a 10-fold increase in sensitivity can be achieved if alkaline phosphatase-labeled streptavidin is used (Dentler, 1992). In addition to labeling membrane proteins prior to SDS-PAGE, putative membrane proteins can be identified by staining blotted proteins with concanavalin A (Con A) and HRP (Pagliaro and Wolfe, 1987). This method is also described below. Although we find this to be an excellent method to detect concanavalin A, an alternative method uses biotinylated concanavalin A coupled with HRP-avidin (R. A. Bloodgood, personal communication). These methods were developed for Tetrahymena cells, but similar methods have been used for Chlamydomonas (Reinhart and Bloodgood, 1988). The method is simple and exceptionally reproducible. Care should be taken to avoid labeling in buffers containing amines that will compete for the ligand (e.g., Tris, azide, glycine, or ammonia). As opposed to other biotinylated compounds, which have half-lives of hours in aqueous solutions, NHS-LC biotin has an extremely short half-life and virtually all labeling is completed within 5- 10 minutes. For all labeling, dissolve the reagent in water or culture medium immediately before use or add solid compound to the medium containing cells.
57. Nonradioactive Membrane Surface Labeling
409
11. Methods A. Labeling Tetrahymena Cilia with NHS-LC Biotin 1. Solution and Material
HNMK: 50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonicacid (Hepes), pH 7.4, 36 mM NaCl, 0.1 mM MgSO,, 1 mM KCl NHS-LC Biotin (Pierce)
2. Procedure 1. Harvest Tetruhymenu and wash three times with HNMK. Use a clinical centrifuge with round-bottom tubes. Centrifuge at setting 5 for 5 minutes. Suspend the cells gently by squirting a jet of buffer from a Pasteur pipet into the pellet and then pulling cells up and down in the pipet as gently as possible. Check for cell breakage by examining the cells with the light microscope. For labeling, suspend cells to approximately lo7 cells/ml. 2. Take one portion of cells and set aside as a control for nonspecific binding of streptavidin to the polypeptides. 3. Make 8 ml of cells 1 mg/ml with NHS-LC biotin. This can be done by adding solid biotin to the solution of cells or by dissolving biotin in a small volume of HNMK. Cells can be labeled in other than HNMK buffers but the buffer must not contain biotinylatable amine groups. Incubate cells in a small beaker on a shaker for 10 minutes and then dilute with 10 vol of HNMK. 4. Pellet cells and wash at least three times with HNMK. 5 . Deciliate cells and purify cilia and membrane fractions as described in Chapters 3 and 55.
Separate proteins by SDS-PAGE. If desired, proteins can initially be separated by isoelectric focusing and then separated by SDS-PAGE. Then blot polypeptides to nitrocellulose following standard procedures (see Dentler, 1992). Stain the biotinylated proteins on the blot using the following procedure. B. Alkaline Phosphatase-Streptavidin Staining of Biotinylated Protein
1. Solutions TBS: 150 m M NaCl, 10 mM Tris, pH 7.5 TBST: TBS + 0.1% Tween 20 TBSAT: TBST + 0.1% bovine serum albumin (BSA) Alkaline phosphatase-labeled streptavidin (Zymed Laboratories, San Francisco, CA) Alkaline phosphatase developer: For 10 ml, mix 66 ml NBT + 33 ml BCIP (see below) + 9.9 ml alkaline phosphatase buffer
410
William L. Dentler
Alkaline phosphatase buffer: 100 mM Tris, pH 9.5, 100 mM NaC1, 5 mM MgClz NBT (nitroblue tetrazolium, Sigma N6876): 50 mg/ml in 70% dimethyl formamide BCIP (5-bromo-4-chloro-3 indoyl-phosphate, Sigma B8503): 50 mg/ml in 100% dimethyl formamide Stop solution: 3% trichloracetic acid (or rinse well with water)
2. Procedure 1. Wash blot twice with TBSAT, 10 minutes per wash. 2. Incubate in alkaline phosphatase-labeled streptavidin in TBSAT for 1-2 hours at room temperature. 3. Wash three times with TBSAT, 10 minutes per wash. 4. Wash three times with TBST, 15 minutes per wash. 5 . Develop with alkaline phosphatase developer. 6. Pour off developer and stop reaction with 3% trichloracetic acid (optional-can just wash with water). 7. Photograph on Kodak Technical Pan film with a yellow filter. Develop film in Kodak HC-110 developer, dilution B. C. Labeling Blotted Proteins with Concanavalin A The key to this procedure is the fact that HRP is a Con A-binding protein (Hawkes, 1982). As a control for nonspecific HRP binding, mix 0.1 M (Y-Dmethylmanoside with Con A to competitively inhibit Con A binding.
1. Solutions
TPBS: PBS + 0.1% BSA (optional, many do not use BSA), 0.1% Tween 20, 1 mM CaC12, 0.1 mM MnClZ, 1 mM MgClZ 50 m M Tris-C1, pH 6.9, with HCl Developer: 20 ml of 0.3% 4-chloro-1-naphthol in 100% methanol HzOz HRP
2. Procedure 1. Blot proteins onto nitrocellulose. 2. Wash three times in TPBS. 3. Incubate with 50 pg/ml Con A in TPBS for 60 minutes.
57. Nonradioactive Membrane Surface Labeling
4. 5. 6. 7. 8. 9.
41 1
Wash three times in TPBS. Incubate with 50 pg/ml HRP in TPBS for 60 minutes. Wash twice with TPBS. Wash three times in 50 mM Tris-HC1, pH 6.9, -3 minutes per wash. Develop with 4-chloro-I-naphthol and H,Oz, for 5-10 minutes. Stop development by rinsing with water. Photograph immediately or store in a dark place.
References Bloodgood, R. A., and Workman, L. J. (1984).A flagellar surface glycoprotein mediating cellsubstrate interaction in Chlamydomonas. Cell Motil. 4, 77-87. Dentler, W. L.(1992).Identification of Tetrahymena ciliary surface proteins labeled with sulfosuccinimidyl 6-(biotinamido)hexanoate and Concanavalin A and fractionated with Triton X-114. J. Protozool. 39, 368-378. Goodloe-Holland, C. M., and Luna, E. J. (1987).Purification and characterization of Dictyocstelium discoideum plasma membranes. Methods Cell Biol. 28, 103-128. Hawkes, R. (1982).Identification of Concanavalin A-binding proteins after sodium dodecyl sulfategel electrophoresis and protein blotting. Anal. Biochern. U3, 193-196. Pagliaro, L.,and Wolfe, J. (1987).Concanavalin A binding induces association of possible matingtype receptors with the cytoskeleton of Tetrahymena. Exp. Cell Res. 168, 138-152. Reinhart, F. D., and Bloodgood, R. A. (1988).Membrane-cytoskeleton interactions in the flagellum: a 240,000Mr surface-exposed glycoprotein is tightly associated with the axoneme in Chlarnydomonas moewusii. J . Cell Sci. 89, 521-531. Thompson, J. A., Law, A. L., and Cunningham, D. D. (1987).Selective radiolabeling ofcell surface proteins to a high specific activity. Biochemistry 26, 743-750. Williams, N.E.,Subbaiah, P. V., and Thompson, G. A., Jr. (1980).Studies of membrane formation in Tetrahymena. The identification of membrane proteins and turnover rates in nongrowing cells. J . Biol. Chem. 2S5,296-303.
Nonradioactive Methods for Labeling and Identifying Membrane Surface Proteins William L. Dentler Department of Physiology and Cell Biology University of Kansas Lawrence, Kansas 66045
I. Introduction 11. Methods
A. Labeling Tetrahymena Cilia with NHS-LC Biotin B. Alkaline Phosphatase-Streptavidin Staining of Biotinylated Protein C . Labeling Blotted Proteins with Concanavalin A References
I. Introduction In addition to motility, cilia provide surfaces on which a variety of cell surface molecules are displayed for cell-cell interactions during mating, chemoreception, and a variety of cellular functions. For a typical biflagellate Chlamydornonus cell, the surface area of the flagellar membrane is approximately 15 pm2, about 0.3% of the total surface area of the cell. For Tetrahymena pyriformis, which contains approximately 600 cilia per cell, the total surface area of the membrane can be greater than 40% of the total cell surface area. At present, we have little understanding of the function and identity of most of the ciliary surface proteins, other than the fact that many are present. In a study of Tetrahymena thermophila ciliary membrane proteins, we identified more than 40 ciliary membrane surface proteins, at least 16 of which also bind concanavalin A (Dentler, 1992). Other than 36- and 50-kDa polypeptides that appear to be associated with a microtubule-membrane bridge, the functions of these proteins remain unknown. METHODS IN CELL BIOLOGY. VOL. 47
Copyright 0 1995 by Academic Press. Inc. All rights of reproduction m any form reserved
407
408
William L. Dentler
Putative membrane surface proteins can be identified by iodination (Bloodgood and Workman, 1984; Williams et al., 1980) followed by resolution of proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography. There is, however, concern about this method due to its potential to label cytoplasmic proteins (Thompson et al., 1987) and the use of 1251, In contrast to iodination, labeling with biotin provides a gentle and nonradioactive method to label surface proteins in a variety of cells (see Goodloe-Holland and Luna, 1987). Biotinylation involves the covalent coupling of biotin to a protein or other molecule and the subsequent binding of biotin to avidin or the more specific streptavidin, with which biotin forms an extremely high affinity bond (with a dissociation constant of -lo-'' M). Avidin or streptavidin can be coupled to alkaline phosphatase or other reagents that produce a colorimetric reaction used to identify biotinylated proteins bound to nitrocellulose. Although streptavidin-gold particles should be useful in identifying biotinylated proteins by electron microscopy, we have had relatively little success in obtaining convincing data using this procedure. A variety of compounds, some of which are reversible, can be used to link biotin to proteins, carbohydrates, and nucleic acids. One of the best sources for these compounds is Pierce Chemical, and the reader is encouraged to consult their catalog for new compounds and labeling procedures. We use sulfosuccinimidyl 6-(biotinamido) hexanoate (NHS-LC biotin), which reacts with primary amines (generally lysine epsilon groups) to form a stable covalent amide bond. Reactions are favored in slightly alkaline media, in which the primary amines are unprotonated. The NHS-LC biotin is water soluble and does not penetrate the ciliary membrane to label axonemal or cytoplasmic proteins (Dentler, 1992). The sensitivity of the reagent depends on the detection methods used after SDS-PAGE and blotting to nitrocellulose. Adequate labeling, comparable to iodination, can be achieved using horseradish peroxidase (HRP)-streptavidin to stain the biotin, but more than a 10-fold increase in sensitivity can be achieved if alkaline phosphatase-labeled streptavidin is used (Dentler, 1992). In addition to labeling membrane proteins prior to SDS-PAGE, putative membrane proteins can be identified by staining blotted proteins with concanavalin A (Con A) and HRP (Pagliaro and Wolfe, 1987). This method is also described below. Although we find this to be an excellent method to detect concanavalin A, an alternative method uses biotinylated concanavalin A coupled with HRP-avidin (R. A. Bloodgood, personal communication). These methods were developed for Tetrahymena cells, but similar methods have been used for Chlamydomonas (Reinhart and Bloodgood, 1988). The method is simple and exceptionally reproducible. Care should be taken to avoid labeling in buffers containing amines that will compete for the ligand (e.g., Tris, azide, glycine, or ammonia). As opposed to other biotinylated compounds, which have half-lives of hours in aqueous solutions, NHS-LC biotin has an extremely short half-life and virtually all labeling is completed within 5- 10 minutes. For all labeling, dissolve the reagent in water or culture medium immediately before use or add solid compound to the medium containing cells.
57. Nonradioactive Membrane Surface Labeling
409
11. Methods A. Labeling Tetrahymena Cilia with NHS-LC Biotin 1. Solution and Material
HNMK: 50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonicacid (Hepes), pH 7.4, 36 mM NaCl, 0.1 mM MgSO,, 1 mM KCl NHS-LC Biotin (Pierce)
2. Procedure 1. Harvest Tetruhymenu and wash three times with HNMK. Use a clinical centrifuge with round-bottom tubes. Centrifuge at setting 5 for 5 minutes. Suspend the cells gently by squirting a jet of buffer from a Pasteur pipet into the pellet and then pulling cells up and down in the pipet as gently as possible. Check for cell breakage by examining the cells with the light microscope. For labeling, suspend cells to approximately lo7 cells/ml. 2. Take one portion of cells and set aside as a control for nonspecific binding of streptavidin to the polypeptides. 3. Make 8 ml of cells 1 mg/ml with NHS-LC biotin. This can be done by adding solid biotin to the solution of cells or by dissolving biotin in a small volume of HNMK. Cells can be labeled in other than HNMK buffers but the buffer must not contain biotinylatable amine groups. Incubate cells in a small beaker on a shaker for 10 minutes and then dilute with 10 vol of HNMK. 4. Pellet cells and wash at least three times with HNMK. 5 . Deciliate cells and purify cilia and membrane fractions as described in Chapters 3 and 55.
Separate proteins by SDS-PAGE. If desired, proteins can initially be separated by isoelectric focusing and then separated by SDS-PAGE. Then blot polypeptides to nitrocellulose following standard procedures (see Dentler, 1992). Stain the biotinylated proteins on the blot using the following procedure. B. Alkaline Phosphatase-Streptavidin Staining of Biotinylated Protein
1. Solutions TBS: 150 m M NaCl, 10 mM Tris, pH 7.5 TBST: TBS + 0.1% Tween 20 TBSAT: TBST + 0.1% bovine serum albumin (BSA) Alkaline phosphatase-labeled streptavidin (Zymed Laboratories, San Francisco, CA) Alkaline phosphatase developer: For 10 ml, mix 66 ml NBT + 33 ml BCIP (see below) + 9.9 ml alkaline phosphatase buffer
410
William L. Dentler
Alkaline phosphatase buffer: 100 mM Tris, pH 9.5, 100 mM NaC1, 5 mM MgClz NBT (nitroblue tetrazolium, Sigma N6876): 50 mg/ml in 70% dimethyl formamide BCIP (5-bromo-4-chloro-3 indoyl-phosphate, Sigma B8503): 50 mg/ml in 100% dimethyl formamide Stop solution: 3% trichloracetic acid (or rinse well with water)
2. Procedure 1. Wash blot twice with TBSAT, 10 minutes per wash. 2. Incubate in alkaline phosphatase-labeled streptavidin in TBSAT for 1-2 hours at room temperature. 3. Wash three times with TBSAT, 10 minutes per wash. 4. Wash three times with TBST, 15 minutes per wash. 5 . Develop with alkaline phosphatase developer. 6. Pour off developer and stop reaction with 3% trichloracetic acid (optional-can just wash with water). 7. Photograph on Kodak Technical Pan film with a yellow filter. Develop film in Kodak HC-110 developer, dilution B. C. Labeling Blotted Proteins with Concanavalin A The key to this procedure is the fact that HRP is a Con A-binding protein (Hawkes, 1982). As a control for nonspecific HRP binding, mix 0.1 M (Y-Dmethylmanoside with Con A to competitively inhibit Con A binding.
1. Solutions
TPBS: PBS + 0.1% BSA (optional, many do not use BSA), 0.1% Tween 20, 1 mM CaC12, 0.1 mM MnClZ, 1 mM MgClZ 50 m M Tris-C1, pH 6.9, with HCl Developer: 20 ml of 0.3% 4-chloro-1-naphthol in 100% methanol HzOz HRP
2. Procedure 1. Blot proteins onto nitrocellulose. 2. Wash three times in TPBS. 3. Incubate with 50 pg/ml Con A in TPBS for 60 minutes.
57. Nonradioactive Membrane Surface Labeling
4. 5. 6. 7. 8. 9.
41 1
Wash three times in TPBS. Incubate with 50 pg/ml HRP in TPBS for 60 minutes. Wash twice with TPBS. Wash three times in 50 mM Tris-HC1, pH 6.9, -3 minutes per wash. Develop with 4-chloro-I-naphthol and H,Oz, for 5-10 minutes. Stop development by rinsing with water. Photograph immediately or store in a dark place.
References Bloodgood, R. A., and Workman, L. J. (1984).A flagellar surface glycoprotein mediating cellsubstrate interaction in Chlamydomonas. Cell Motil. 4, 77-87. Dentler, W. L.(1992).Identification of Tetrahymena ciliary surface proteins labeled with sulfosuccinimidyl 6-(biotinamido)hexanoate and Concanavalin A and fractionated with Triton X-114. J. Protozool. 39, 368-378. Goodloe-Holland, C. M., and Luna, E. J. (1987).Purification and characterization of Dictyocstelium discoideum plasma membranes. Methods Cell Biol. 28, 103-128. Hawkes, R. (1982).Identification of Concanavalin A-binding proteins after sodium dodecyl sulfategel electrophoresis and protein blotting. Anal. Biochern. U3, 193-196. Pagliaro, L.,and Wolfe, J. (1987).Concanavalin A binding induces association of possible matingtype receptors with the cytoskeleton of Tetrahymena. Exp. Cell Res. 168, 138-152. Reinhart, F. D., and Bloodgood, R. A. (1988).Membrane-cytoskeleton interactions in the flagellum: a 240,000Mr surface-exposed glycoprotein is tightly associated with the axoneme in Chlarnydomonas moewusii. J . Cell Sci. 89, 521-531. Thompson, J. A., Law, A. L., and Cunningham, D. D. (1987).Selective radiolabeling ofcell surface proteins to a high specific activity. Biochemistry 26, 743-750. Williams, N.E.,Subbaiah, P. V., and Thompson, G. A., Jr. (1980).Studies of membrane formation in Tetrahymena. The identification of membrane proteins and turnover rates in nongrowing cells. J . Biol. Chem. 2S5,296-303.