- Email: [email protected]
[52] Gel overlay methods for detecting specific protein-protein interactions
[52]
GEL OVERLAY METHODS
555
about 30 min at 20,000 rpm in a Beckman Type 40 rotor. We have not carried out such experiments. Other Methods
Pollard and Cooper ~ describe methods for studying actin filament networks, which could also be used to study the microtubule-neurofilament network. These include a test tube inversion assay, low-speed sedimentation, yield-strength measurements with the falling-ball device, and measurements with a gelmeter, rotational viscometers, micromagnetorheometer, and viscoelastometers. Other possibly useful methods include zonal centrifugation through preformed density gradients, and quick-freeze deep-etch electron microscopy. 15 Chemical cross-linkers might be useful for determining the relative position of the proteins in the network. Acknowledgments The authors are supported by Research Grants GM29834 (to R. C. W.) and NS07738 (to E. J. A.) from the National Institutes of Health, and by the Vanderbilt University Research Council. We thank Drs. Stephanie M. Jones, John J. Correia, and Marschall S. Runge for helpful discussion. 15 j. E. Heuser and M. W. Kirschner, J. Cell Biol. 86, 212 (1980).
[52] G e l O v e r l a y M e t h o d s for D e t e c t i n g Specific Protein-Protein Interactions By JOANN J. OTTO
Gel overlays provide a useful method to detect protein-protein interactions in oitro. Proteins which were not previously known to interact can be discovered by this method. In a gel overlay, samples of proteins are separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the proteins are at least partially renatured in the gel. The gel is then immersed in a radiolabeled protein. After washing to remove nonspecifically absorbed label, the gel is autoradiographed to detect peptides to which the radiolabeled protein has bound. With a gel overlay, the molecular weight of an interacting protein is known, only METHODS IN ENZYMOLOGY, VOL. 134
Copyright © 1986by Academic Press, Inc. All rights of reproduction in any form reserved.
556
ANALYSIS OF CYTOSKELETAL PROTEINS
[52]
microgram amounts of the proteins are needed for the gel electrophoresis, protein purification schemes can be readily assayed, and binding to proteins which are only soluble under denaturing conditions can be detected. This type of assay is limited as follows: binding proteins which do not renature after SDS-PAGE will not be detected, binding proteins consisting of more than one subunit will not be observed if the intact molecule is required for binding, and there is the possibility that labeling of the protein probe by iodination will cause it to lose its ability to interact. As in the case with any assay, it is desirable and necessary to confirm observations of protein interaction found in a gel overlay with other nonrelated methods of detecting protein-protein interactions. In designing a gel overlay for a particular protein, the more that is known about the chemistry of the protein the easier the design will be. For example, if the protein is sensitive to calcium, one can take advantage of this to allow binding in the presence or absence of calcium; if a protein is most active at a particular pH, this pH can be used for the assay. Ideally, the activity of the iodinated protein should be similar to that of the native protein and this should be determined if there is a way in which the protein can be assayed (e.g., enzymatic activity). Common to all gel overlays are the following features: fixation of the proteins in place in the gel, removal of SDS from the gel by washing with ethanol, renaturation of the proteins in the gel in a suitable buffer, incubation of the gel with the iodinated probe, extensive washing of the gel, and autoradiography. The buffer for the overlay should contain at least one protein to block nonspecific binding sites in the gel. In our experience with 125I-labeled vinculin and 125I-labeled talin gel overlays, the combination of gelatin and bovine serum albumin (BSA) works well to inhibit nonspecific binding; gelatin alone is insufficient. Background binding is further reduced by the inclusion of a nonionic detergent such as Triton X-100 or Nonidet P-40. The addition of either 2-mercaptoethanol or dithiothreitol prevents interaction of the iodinated probe and proteins in the gel by disulfide bonds. Since most gel overlay methods involve incubation of the gel for at least several days, sodium azide should be included in the buffer to inhibit bacterial growth. Finally, we have found that shaking the gel constantly throughout the procedure greatly reduces the nonspecific background on the autoradiograph. Below I describe three specific procedures for gel overlays for four different proteins: vinculin, talin, actin, and calmodulin. Development of gel overlays for other proteins will probably require modification of one of these procedures, taking into account the considerations outlined above.
[52]
GEL OVERLAY METHODS
557
125I-Labeled Vinculin and 125I-Labeled Talin Gel Overlay This gel overlay method was originally described for vinculin by Otto'; in addition, the same procedure also works for talin. 2 Using this method on samples of chick embryo fibroblast proteinsJ25I-labeled vinculin was observed to bind to proteins of 220 kDa, subsequently identified as talin, 3 and 130 kDa, identified as vinculin. ~ ~25I-labeled talin binds only to vinculin in gels of chick fibroblast proteins.2 Burridge and Mangeat 3 have used a slightly different gel overlay method based on Burridge's 4 gel overlay method for lectins and antibodies and have obtained similar results except that they do not detect vinculin-vinculin binding. It is not clear whether this difference is due to use of a different iodination method for vinculin (the chloramine-T method 5) or to different solution conditions. Here I describe only the methods which we use. Vinculin is purified according to a modification6 of the method of Feramisco and Burridge. 7 If low-molecular-weight contaminants are evident in the vinculin preparation, the protein is further purified according to Evans e t al. 8 Talin is purified according to Burridge and Connell. 9 For use in the overlay, vinculin or talin can be iodinated either with the Bolton-Hunter reagent or by the Iodobeads procedure (Pierce Chemical Co.). To iodinate with the Bolton-Hunter reagent, the protein is dialyzed extensively against 0.125 M sodium borate buffer, pH 8.4. Then 25-50/zg of the protein in 25-50/xl is incubated for 30 min at 4° with 2 mCi ~25Ilabeled Bolton-Hunter reagent that has been dried under a gentle stream of nitrogen. The reaction is stopped by the addition of 200/xl 0.2 M glycine in 0.125 M sodium borate, pH 8.4, for 15 min at 4°. Iodinated protein is separated from unreacted Bolton-Hunter reagent by chromatography on Sephadex G-25 equilibrated with 0.1 M NaCI, 0.05% NAN3, 0.25% gelatin, and 0.05 M phosphate buffer, pH 7.5. The specific activity of ~z5I-labeled vinculin from this procedure is 3-8 /zCi//~g. To iodinate vinculin or talin using the Iodobeads procedure, the protein is dialyzed against 0.05 M sodium phosphate buffer, pH 7.2. The Iodobeads (we use three beads for 1 ml of vinculin at 0.6 mg/ml) are washed twice with 1 ml J. J. Otto, J. Cell Biol. 97, 1283 (1983). 2 W. Toole and J. J. Otto, unpublished observation (1984). K. Burridge and P. Mangeat, Nature (London) 305, 744 (1984). 4 K. Burridge, Proc. Natl. Acad. Sci. U.S.A. 73, 4457 (1976). 5 W. M. Hunter and F. C. Greenwood, Nature (London) 194, 495 (1962). 6 j. j. Otto, Cell Mot. Cytoskel. 6, 48 (1986). 7 j. R. Feramisco and K. Burridge, J. Biol. Chem. 255, 1194 (1980). 8 R. R. Evans, R. M. Robson, and M. H. Stromer, J. Biol. Chem. 259, 3916 (1984). 9 K. Burridge and L. ConneU, J. Cell Biol. 97, 359 (1983).
558
ANALYSIS OF CYTOSKELETAL PROTEINS
[52]
of the phosphate buffer and then dried on a piece of filter paper. Na~25I (12 mCi) is placed in a microfuge tube and 200 tzl 0.05 M sodium phosphate buffer is added. The beads are then added to the 1251solution. After 5 min, 1 ml of the protein solution is added and the reaction allowed to proceed for 15 min with the tube being rocked on a small test tube rocking platform. The solution is then chromatographed on Sephadex G-25 as describe above to separate the protein from unreacted Na~25I. After iodination of vinculin either with the Bolton-Hunter reagent or the Iodobeads procedure, the iodinated vinculin is diluted to 12/.~Ci/ml with the column buffer and 2-mercaptoethanol is added to the vinculin solution to 15 mM. We have found that if the iodinated vinculin is stored at a higher specific activity, it loses its ability to bind in the gel overlay assay. The 2-mercaptoethanol appears to prevent aggregation of the protein. Samples of proteins to be assayed are separated by SDS-PAGE. The proteins are fixed in the gels with 40% methanol, 10% acetic acid in distilled water for 30 min. The gels are then rinsed with three changes of water and the SDS is removed by washing the gel with 10% ethanol for at least 4 hr or as long as overnight with one solution change. The gels are then equilibrated with overlay buffer: 0.5% BSA, 0.25% gelatin, I% Nonidet P-40, 0.01 M NaCI, 0.05% NAN3, I mM EGTA, 15 mM 2-mercaptoethanol, and 20 mM HEPES, pH 7.5. This equilibration and the washes after incubation with ~25I-labeled vinculin are done in glass baking dishes with constant shaking on a reciprocating shaker. The gels are incubated with I-2/~Ci 125I-labeled vinculin/ml in the same buffer for 24 hr at 37°. For this incubation, the gel is sealed in a Sears Seal n' Save polyethylene bag and rocked on a small test tube rocking platform. After washing with at least 10 changes of the buffer over a three day period, the gel is dried and autoradiographed on Kodak X-Omat AR film (XAR-5) for 6-8 days. All steps are done at room temperature except the incubation with ~25Ilabeled vinculin. For a control, heating (100 ° for 5 min) the iodinated vinculin or talin should eliminate binding. In the case of vinculin, competing for binding with unlabeled vinculin does not reduce the binding of ~25I-labeled vinculin, apparently because vinculin self-associates. 12SI-Labeled Actin Gel Overlay An 125I-labeled actin gel overlay has been described by Snabes et al. l0 With this assay, actin-binding proteins of 40 and 90 kDa were detected in ~oM. C. Snabes, A. E. Boyd, III, and J. Bryan, J. Cell Biol. 90, 809 (1981).
[521
GEL OVERLAY METHODS
559
platelets. The 90-kDa protein requires Ca 2÷ for binding; when EGTA is included in the buffer, binding is eliminated. Actin, purified from rabbit muscle according to Spudich and Watt H followed by gel filtration on Sephacryl S-200, is iodinated with the Bolton-Hunter reagent when it is monomeric (G-actin). The gel containing the samples to be assayed is fixed at room temperature with 40% methanol, 10% acetic acid for 30 min and is then washed for 12 hr with 10% ethanol. Proteins within the gel are renatured for 90-120 min with 0.2 mM CaCI2, 0.2 mM ATP, 0.05% NAN3, 0.1% gelatin, 5.0 mM NaH2PO4, 5.0 mM Na2HPO4, and 0.2 M NaCI, pH 7.4. The gel is sealed into a polyethylene bag and incubated for 12-I6 hr at 4° with 1/zCi 125I-labeled actin/ml. The gel is washed over a 2-day period with at least fives changes of buffer (250 ml/wash) and then dried and autoradiographed. Controls for this assay include using heated iodinated actin to eliminate binding and competing for binding with unlabeled actin to reduce or eliminate binding of labeled actin. 12SI-Labeled Calmodulin Gel Overlay Two different z25I-labeled calmodulin gel overlays have been described by Glenney and Weber lz and Carlin et al.13,14 Both these methods detected the same cytoskeletal calmodulin-binding proteins in the intestinal brush border. Calcium-sensitive calmodulin binding to proteins of 250-280 and 140 kDa and calcium-insensitive binding to a protein of 110 kDa were observed) 2,15 In addition, Howe et al., 15 using the method of Carlin et a l . / 3 detected calcium-sensitive binding to cytoskeletal proteins of 190, 180, and 150 kDa which was not seen by Glenney and Weber) z In the method described by Glenney and Weber,12 calmodulin, purified according to Anderson et al.,16 is iodinated using the Bolton-Hunter reagent. The gel with the samples to be assayed is fixed in 40% methanol, 10% acetic acid for 30 min. It is then rinsed briefly in distilled water and washed for 2 hr or overnight in 10% ethanol to remove the SDS. The gel is incubated in 0.1 M imidazole for 10 min followed by 10 min in the overlay buffer consisting of 20 mM imidazole, 0.2 M KC1, 0.5% gelatin, 0.02% 11 j. A. Spudich and S. Watt, J. Biol. Chem. 246, 4866 (1971). ~2j. Glenney, Jr. and K. Weber, J. Biol. Chem. 255, 10551 (1980). 13 R. K. Carlin, D. J. Grab, and P. Siekowtiz, Ann. N.Y. Acad. Sci. 356, 73 (1980). t4 R. K. Carlin, D. J. Grab, and P. Siekowitz, J. Cell Biol. 89, 449 (1981). i5 C. L. Howe, T. C. S. Keller, III, M. S. Mooseker, and R. H. Wasserman, Proc. Natl. Acad. Sci. U.S.A. 79, 1134 (1982). 16 B. Anderson, M. Osborn, and K. Weber, Cytobiologie 17, 354 (1978).
560
ANALYSIS OF CYTOSKELETALPROTEINS
[52]
•NAN3, pH 7.0 containing either 1 mM CaCI2, 1 mM EGTA, or 1 mM CaCl2 plus 0.1 M chlorpromazine for 10 min. The gel is then incubated for 4-8 hr with 2.5/~Ci 125I-labeled calmodulin/ml at 37° after which it is washed in a large volume of the overlay buffer (five changes over a 2-day period). The gel is then dried and autoradiographed. All incubations are done with shaking and at room temperature, except as noted. As a control all binding should be abolished by incubating in the presence of 0. I mM chlorpromazine and 1 m M CaCI2. Cadin et aL ia use purified calmodulin 17 which is iodinated using the lactoperoxidase procedure for their 125I-labeled calmodulin gel overlay; this calmodulin is still able to activate cyclic nucleotide phosphodiesterase. They note that iodination of calmodulin by the chloramine-T method 5 inactivates the protein. The gel is fixed with 25% isopropanol, 10% acetic acid for 12 hr with at least four solution changes and with shaking. After a 5-10 min wash with distilled water, the gel is incubated for 12 hr with 50 mM Tris, 0.2 M NaC1, and 1 mM CaC12, pH 7.6, with at least four solution changes followed by equilibration with the buffer containing I mg BSA/ml for 2 hr. The authors note that the addition of BSA in this manner immediately before incubation with the iodinated calmodulin increased specific binding at least l0 times. The gel is then placed in a polyethylene bag and incubated with 125I-labeled calmodulin (1/zg/ml) in the buffer without BSA for 12 hr after which the gel is washed for 36 hr with at least six changes of buffer. The gels are stained for 2 hr in 0.25% Coomassie blue in 50% methanol, 7% acetic acid and destained with 25% methanol, 7% acetic acid. The gel is then dried and autoradiographed. Staining the gel permits a direct comparison between stained protein bands and bands which have bound 125I-labeled calmodulin. Acknowledgment This work is supportedby Grant CD-108from the AmericanCancer Society.
17p. G. Richman and C. B. Klee, J. Biol. Chem. 253, 6323 (1978).
GEL OVERLAY METHODS
555
about 30 min at 20,000 rpm in a Beckman Type 40 rotor. We have not carried out such experiments. Other Methods
Pollard and Cooper ~ describe methods for studying actin filament networks, which could also be used to study the microtubule-neurofilament network. These include a test tube inversion assay, low-speed sedimentation, yield-strength measurements with the falling-ball device, and measurements with a gelmeter, rotational viscometers, micromagnetorheometer, and viscoelastometers. Other possibly useful methods include zonal centrifugation through preformed density gradients, and quick-freeze deep-etch electron microscopy. 15 Chemical cross-linkers might be useful for determining the relative position of the proteins in the network. Acknowledgments The authors are supported by Research Grants GM29834 (to R. C. W.) and NS07738 (to E. J. A.) from the National Institutes of Health, and by the Vanderbilt University Research Council. We thank Drs. Stephanie M. Jones, John J. Correia, and Marschall S. Runge for helpful discussion. 15 j. E. Heuser and M. W. Kirschner, J. Cell Biol. 86, 212 (1980).
[52] G e l O v e r l a y M e t h o d s for D e t e c t i n g Specific Protein-Protein Interactions By JOANN J. OTTO
Gel overlays provide a useful method to detect protein-protein interactions in oitro. Proteins which were not previously known to interact can be discovered by this method. In a gel overlay, samples of proteins are separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the proteins are at least partially renatured in the gel. The gel is then immersed in a radiolabeled protein. After washing to remove nonspecifically absorbed label, the gel is autoradiographed to detect peptides to which the radiolabeled protein has bound. With a gel overlay, the molecular weight of an interacting protein is known, only METHODS IN ENZYMOLOGY, VOL. 134
Copyright © 1986by Academic Press, Inc. All rights of reproduction in any form reserved.
556
ANALYSIS OF CYTOSKELETAL PROTEINS
[52]
microgram amounts of the proteins are needed for the gel electrophoresis, protein purification schemes can be readily assayed, and binding to proteins which are only soluble under denaturing conditions can be detected. This type of assay is limited as follows: binding proteins which do not renature after SDS-PAGE will not be detected, binding proteins consisting of more than one subunit will not be observed if the intact molecule is required for binding, and there is the possibility that labeling of the protein probe by iodination will cause it to lose its ability to interact. As in the case with any assay, it is desirable and necessary to confirm observations of protein interaction found in a gel overlay with other nonrelated methods of detecting protein-protein interactions. In designing a gel overlay for a particular protein, the more that is known about the chemistry of the protein the easier the design will be. For example, if the protein is sensitive to calcium, one can take advantage of this to allow binding in the presence or absence of calcium; if a protein is most active at a particular pH, this pH can be used for the assay. Ideally, the activity of the iodinated protein should be similar to that of the native protein and this should be determined if there is a way in which the protein can be assayed (e.g., enzymatic activity). Common to all gel overlays are the following features: fixation of the proteins in place in the gel, removal of SDS from the gel by washing with ethanol, renaturation of the proteins in the gel in a suitable buffer, incubation of the gel with the iodinated probe, extensive washing of the gel, and autoradiography. The buffer for the overlay should contain at least one protein to block nonspecific binding sites in the gel. In our experience with 125I-labeled vinculin and 125I-labeled talin gel overlays, the combination of gelatin and bovine serum albumin (BSA) works well to inhibit nonspecific binding; gelatin alone is insufficient. Background binding is further reduced by the inclusion of a nonionic detergent such as Triton X-100 or Nonidet P-40. The addition of either 2-mercaptoethanol or dithiothreitol prevents interaction of the iodinated probe and proteins in the gel by disulfide bonds. Since most gel overlay methods involve incubation of the gel for at least several days, sodium azide should be included in the buffer to inhibit bacterial growth. Finally, we have found that shaking the gel constantly throughout the procedure greatly reduces the nonspecific background on the autoradiograph. Below I describe three specific procedures for gel overlays for four different proteins: vinculin, talin, actin, and calmodulin. Development of gel overlays for other proteins will probably require modification of one of these procedures, taking into account the considerations outlined above.
[52]
GEL OVERLAY METHODS
557
125I-Labeled Vinculin and 125I-Labeled Talin Gel Overlay This gel overlay method was originally described for vinculin by Otto'; in addition, the same procedure also works for talin. 2 Using this method on samples of chick embryo fibroblast proteinsJ25I-labeled vinculin was observed to bind to proteins of 220 kDa, subsequently identified as talin, 3 and 130 kDa, identified as vinculin. ~ ~25I-labeled talin binds only to vinculin in gels of chick fibroblast proteins.2 Burridge and Mangeat 3 have used a slightly different gel overlay method based on Burridge's 4 gel overlay method for lectins and antibodies and have obtained similar results except that they do not detect vinculin-vinculin binding. It is not clear whether this difference is due to use of a different iodination method for vinculin (the chloramine-T method 5) or to different solution conditions. Here I describe only the methods which we use. Vinculin is purified according to a modification6 of the method of Feramisco and Burridge. 7 If low-molecular-weight contaminants are evident in the vinculin preparation, the protein is further purified according to Evans e t al. 8 Talin is purified according to Burridge and Connell. 9 For use in the overlay, vinculin or talin can be iodinated either with the Bolton-Hunter reagent or by the Iodobeads procedure (Pierce Chemical Co.). To iodinate with the Bolton-Hunter reagent, the protein is dialyzed extensively against 0.125 M sodium borate buffer, pH 8.4. Then 25-50/zg of the protein in 25-50/xl is incubated for 30 min at 4° with 2 mCi ~25Ilabeled Bolton-Hunter reagent that has been dried under a gentle stream of nitrogen. The reaction is stopped by the addition of 200/xl 0.2 M glycine in 0.125 M sodium borate, pH 8.4, for 15 min at 4°. Iodinated protein is separated from unreacted Bolton-Hunter reagent by chromatography on Sephadex G-25 equilibrated with 0.1 M NaCI, 0.05% NAN3, 0.25% gelatin, and 0.05 M phosphate buffer, pH 7.5. The specific activity of ~z5I-labeled vinculin from this procedure is 3-8 /zCi//~g. To iodinate vinculin or talin using the Iodobeads procedure, the protein is dialyzed against 0.05 M sodium phosphate buffer, pH 7.2. The Iodobeads (we use three beads for 1 ml of vinculin at 0.6 mg/ml) are washed twice with 1 ml J. J. Otto, J. Cell Biol. 97, 1283 (1983). 2 W. Toole and J. J. Otto, unpublished observation (1984). K. Burridge and P. Mangeat, Nature (London) 305, 744 (1984). 4 K. Burridge, Proc. Natl. Acad. Sci. U.S.A. 73, 4457 (1976). 5 W. M. Hunter and F. C. Greenwood, Nature (London) 194, 495 (1962). 6 j. j. Otto, Cell Mot. Cytoskel. 6, 48 (1986). 7 j. R. Feramisco and K. Burridge, J. Biol. Chem. 255, 1194 (1980). 8 R. R. Evans, R. M. Robson, and M. H. Stromer, J. Biol. Chem. 259, 3916 (1984). 9 K. Burridge and L. ConneU, J. Cell Biol. 97, 359 (1983).
558
ANALYSIS OF CYTOSKELETAL PROTEINS
[52]
of the phosphate buffer and then dried on a piece of filter paper. Na~25I (12 mCi) is placed in a microfuge tube and 200 tzl 0.05 M sodium phosphate buffer is added. The beads are then added to the 1251solution. After 5 min, 1 ml of the protein solution is added and the reaction allowed to proceed for 15 min with the tube being rocked on a small test tube rocking platform. The solution is then chromatographed on Sephadex G-25 as describe above to separate the protein from unreacted Na~25I. After iodination of vinculin either with the Bolton-Hunter reagent or the Iodobeads procedure, the iodinated vinculin is diluted to 12/.~Ci/ml with the column buffer and 2-mercaptoethanol is added to the vinculin solution to 15 mM. We have found that if the iodinated vinculin is stored at a higher specific activity, it loses its ability to bind in the gel overlay assay. The 2-mercaptoethanol appears to prevent aggregation of the protein. Samples of proteins to be assayed are separated by SDS-PAGE. The proteins are fixed in the gels with 40% methanol, 10% acetic acid in distilled water for 30 min. The gels are then rinsed with three changes of water and the SDS is removed by washing the gel with 10% ethanol for at least 4 hr or as long as overnight with one solution change. The gels are then equilibrated with overlay buffer: 0.5% BSA, 0.25% gelatin, I% Nonidet P-40, 0.01 M NaCI, 0.05% NAN3, I mM EGTA, 15 mM 2-mercaptoethanol, and 20 mM HEPES, pH 7.5. This equilibration and the washes after incubation with ~25I-labeled vinculin are done in glass baking dishes with constant shaking on a reciprocating shaker. The gels are incubated with I-2/~Ci 125I-labeled vinculin/ml in the same buffer for 24 hr at 37°. For this incubation, the gel is sealed in a Sears Seal n' Save polyethylene bag and rocked on a small test tube rocking platform. After washing with at least 10 changes of the buffer over a three day period, the gel is dried and autoradiographed on Kodak X-Omat AR film (XAR-5) for 6-8 days. All steps are done at room temperature except the incubation with ~25Ilabeled vinculin. For a control, heating (100 ° for 5 min) the iodinated vinculin or talin should eliminate binding. In the case of vinculin, competing for binding with unlabeled vinculin does not reduce the binding of ~25I-labeled vinculin, apparently because vinculin self-associates. 12SI-Labeled Actin Gel Overlay An 125I-labeled actin gel overlay has been described by Snabes et al. l0 With this assay, actin-binding proteins of 40 and 90 kDa were detected in ~oM. C. Snabes, A. E. Boyd, III, and J. Bryan, J. Cell Biol. 90, 809 (1981).
[521
GEL OVERLAY METHODS
559
platelets. The 90-kDa protein requires Ca 2÷ for binding; when EGTA is included in the buffer, binding is eliminated. Actin, purified from rabbit muscle according to Spudich and Watt H followed by gel filtration on Sephacryl S-200, is iodinated with the Bolton-Hunter reagent when it is monomeric (G-actin). The gel containing the samples to be assayed is fixed at room temperature with 40% methanol, 10% acetic acid for 30 min and is then washed for 12 hr with 10% ethanol. Proteins within the gel are renatured for 90-120 min with 0.2 mM CaCI2, 0.2 mM ATP, 0.05% NAN3, 0.1% gelatin, 5.0 mM NaH2PO4, 5.0 mM Na2HPO4, and 0.2 M NaCI, pH 7.4. The gel is sealed into a polyethylene bag and incubated for 12-I6 hr at 4° with 1/zCi 125I-labeled actin/ml. The gel is washed over a 2-day period with at least fives changes of buffer (250 ml/wash) and then dried and autoradiographed. Controls for this assay include using heated iodinated actin to eliminate binding and competing for binding with unlabeled actin to reduce or eliminate binding of labeled actin. 12SI-Labeled Calmodulin Gel Overlay Two different z25I-labeled calmodulin gel overlays have been described by Glenney and Weber lz and Carlin et al.13,14 Both these methods detected the same cytoskeletal calmodulin-binding proteins in the intestinal brush border. Calcium-sensitive calmodulin binding to proteins of 250-280 and 140 kDa and calcium-insensitive binding to a protein of 110 kDa were observed) 2,15 In addition, Howe et al., 15 using the method of Carlin et a l . / 3 detected calcium-sensitive binding to cytoskeletal proteins of 190, 180, and 150 kDa which was not seen by Glenney and Weber) z In the method described by Glenney and Weber,12 calmodulin, purified according to Anderson et al.,16 is iodinated using the Bolton-Hunter reagent. The gel with the samples to be assayed is fixed in 40% methanol, 10% acetic acid for 30 min. It is then rinsed briefly in distilled water and washed for 2 hr or overnight in 10% ethanol to remove the SDS. The gel is incubated in 0.1 M imidazole for 10 min followed by 10 min in the overlay buffer consisting of 20 mM imidazole, 0.2 M KC1, 0.5% gelatin, 0.02% 11 j. A. Spudich and S. Watt, J. Biol. Chem. 246, 4866 (1971). ~2j. Glenney, Jr. and K. Weber, J. Biol. Chem. 255, 10551 (1980). 13 R. K. Carlin, D. J. Grab, and P. Siekowtiz, Ann. N.Y. Acad. Sci. 356, 73 (1980). t4 R. K. Carlin, D. J. Grab, and P. Siekowitz, J. Cell Biol. 89, 449 (1981). i5 C. L. Howe, T. C. S. Keller, III, M. S. Mooseker, and R. H. Wasserman, Proc. Natl. Acad. Sci. U.S.A. 79, 1134 (1982). 16 B. Anderson, M. Osborn, and K. Weber, Cytobiologie 17, 354 (1978).
560
ANALYSIS OF CYTOSKELETALPROTEINS
[52]
•NAN3, pH 7.0 containing either 1 mM CaCI2, 1 mM EGTA, or 1 mM CaCl2 plus 0.1 M chlorpromazine for 10 min. The gel is then incubated for 4-8 hr with 2.5/~Ci 125I-labeled calmodulin/ml at 37° after which it is washed in a large volume of the overlay buffer (five changes over a 2-day period). The gel is then dried and autoradiographed. All incubations are done with shaking and at room temperature, except as noted. As a control all binding should be abolished by incubating in the presence of 0. I mM chlorpromazine and 1 m M CaCI2. Cadin et aL ia use purified calmodulin 17 which is iodinated using the lactoperoxidase procedure for their 125I-labeled calmodulin gel overlay; this calmodulin is still able to activate cyclic nucleotide phosphodiesterase. They note that iodination of calmodulin by the chloramine-T method 5 inactivates the protein. The gel is fixed with 25% isopropanol, 10% acetic acid for 12 hr with at least four solution changes and with shaking. After a 5-10 min wash with distilled water, the gel is incubated for 12 hr with 50 mM Tris, 0.2 M NaC1, and 1 mM CaC12, pH 7.6, with at least four solution changes followed by equilibration with the buffer containing I mg BSA/ml for 2 hr. The authors note that the addition of BSA in this manner immediately before incubation with the iodinated calmodulin increased specific binding at least l0 times. The gel is then placed in a polyethylene bag and incubated with 125I-labeled calmodulin (1/zg/ml) in the buffer without BSA for 12 hr after which the gel is washed for 36 hr with at least six changes of buffer. The gels are stained for 2 hr in 0.25% Coomassie blue in 50% methanol, 7% acetic acid and destained with 25% methanol, 7% acetic acid. The gel is then dried and autoradiographed. Staining the gel permits a direct comparison between stained protein bands and bands which have bound 125I-labeled calmodulin. Acknowledgment This work is supportedby Grant CD-108from the AmericanCancer Society.
17p. G. Richman and C. B. Klee, J. Biol. Chem. 253, 6323 (1978).