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silver) and complete protein staining (AuroDye) on Western blots
Journal of Immunological Methods, 89 (1986) 89-91
89
Elsevier JIM 03888
Sequential immunostaining (gold/silver) and complete protein staining (AuroDye) on Western blots Guy Daneels, Marc Moeremans, Marc De Raeymaeker and Jan De Mey Laboratory of Biochemical Cytology, Division of Cellular Biology and Chemotherapy, Department of Life Sciences. Janssen Pharmaceutica Research Laboratories, B-2340 Beerse, Belgium
(Received 20 November 1985, accepted 11 December 1985)
A double staining method is described which combines immunodetection with sensitive staining of the complete electropherogram on the same membrane. The method is based on the use of Tween 20 as blocking agent, and uses immunogold/silver staining of specific antigens and gold staining of the overall protein pattern with AuroDye. This double staining makes possible the exact location of an immunodetected band within a complex protein pattern. Key words: Colloidal gold," Colloidal iron," lmmunogold/silver staining," Protein blotting," Immunoblotting," Electrophoresis
Introduction
Overlay techniques for electrophoretically separated proteins transferred to immobilizing membranes have become important tools for the analysis of binding activities of different kinds of proteins, especially antibodies (for review see Gershoni and Palade, 1983). In order to make a comparison between an immunodetected band and the total electropherogram one blot or blot unit is usually immunostained and a duplicate stained for the overall protein pattern. Recently we have shown that colloidal gold particles linked to secondary antibodies (Moeremans et al., 1984) and colloidal metal particles (Moeremans et al., 1985, 1986) can be used for sensitive immunostaining and complete protein staining, respectively. Because the preparation of a duplicate blot may sometimes be difficult and is of course both time and sample Abbreviations: PBS-TW, phosphate-buffered saline supplemented with Tween 20; 1D and 2D: one- and two-dimensional gel electrophoresis; NEPHGE: non-equilibrium pH gradient electrophoresis; SDS: sodium dodecyl sulphate.
consuming, especially for 2D blots, we have developed a method that allows for double staining on the same blot. It is based on the observation (Batteiger et al., 1982) that the use of Tween 20 as blocking agent makes it possible to stain the overall protein pattern after specific probing with 125Ilabelled antibodies and before autoradiography. The method presented here has high sensitivity for both immunodetection and complete protein staining. It uses immunogold/silver staining followed by gold staining of the overall protein pattern.
Materials and methods
All reagents were of analytical grade quality and double distilled water was used throughout. A whole cell extract of rat hepatoma cells (HTC) was prepared for 2D electrophoresis according to Garrels (1979). After N E P H G E in the first dimension, proteins were separated according to their molecular weight by SDS electrophoresis (Blattler et al., 1972) and the separated proteins were elec-
0022-1759/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
90 trotransferred to a nitrocellulose membrane (Towbin et al., 1979). Care was taken to place an additional membrane at the cathodic side in the sandwich to adsorb impurities from the blotting system. After transfer, the blot was saturated with PBS (without Ca 2+ and Mg 2+, Gibco), pH 7.2, containing 0.3% Tween 20 (polyoxyethylene sorbitan monolaureate, Bio-Rad), PBS-TW, for 30 min at 37°C and washed 3 x 10 min with PBS-TW at room temperature. All subsequent steps were
!
A
carried out at room temperature. The blot was incubated with a rabbit antibody directed against dog brain tubulin. The antibody was diluted in PBS-0.05% Tween 20 to a concentration of 1 /~g/ml. After incubation for 2 h, the blot was washed (3 x 5 min) with PBS-0.05% TW and the specific interaction was visualized by incubation in goat anti-rabbit globulin linked to 20 nm gold particles (AuroProbe BL GAR, Janssen Life Sciences Products, Beerse, Belgium) for 2 h. This immunogold probe was diluted with PBS-0.05% TW to an optical density at 520 nm of 0.05. After washing in PBS-0.05% TW (3 × 5 min) and in double distilled water (3 × 5 min) this signal was silver-enhanced and fixed (Moeremans et al., 1984) using the IntenSE silver enhancement kit (Janssen Life Sciences Products, Beerse, Belgium). The blot was washed (3 × 5 min) with a large excess of distilled water and a picture was taken. Before processing for gold staining with AuroDye (Janssen Life Sciences Products, Beerse, Belgium) the blot was incubated for 5 rain in PBS 0.3% Tween 20. Then the blot was incubated overnight in the gold stain. After rinsing in excess water the blot was air-dried.
Results In order to demonstrate the utility of this method a whole cell extract of rat hepatoma cells was prepared and run on a 2D gel. After electrotransfer, the nitrocellulose membrane was saturated with Tween 20. The use of Tween 20 as blocking agent did not influence the subsequent immunostaining (Fig. 1A). As shown in Fig. 1B the subsequent protein staining with AuroDye was also not affected by the preceding incubation steps with PBS-TW and the immunogold/silver staining. It showed the high sensitivity as could be expected from this colloidal gold stain.
m
Discussion
Q
B Fig. 1. lmmunodetection (indirect immunogold/silverstaining) of tubulin (arrow) with rabbit anti-tubulin (A). Sequential overall protein staining of the same blot with AuroDye (B).
Using Tween 20 as a blocking agent (Batteiger et al., 1982) one can perform sequential imm u n o a u t o r a d i o g r a p h y and complete protein staining on the same blot. The results presented
91
here show that this can be done with high, matching sensitivity, both detection methods generating stains on the blot. The colloidal iron stain (FerriDye, Janssen Life Sciences Products, Beerse, Belgium) can also be used as a complete protein stain (results not shown). Other overlay techniques (using, for example, lectins) and detection systems can probably be used in the specific probing step. The high contrast provided by the silver enhancement is however a distinct advantage. It is sometimes difficult to distinguish weak immunodetected bands within the complete protein pattern. This problem is easily overcome by taking a picture before complete protein staining. In general the complete protein staining is not only helpful for defining the exact localization of selectively stained components, but also as a quality control of the protein transfer. In our hands, the use of Tween 20, a detergent, did not influence the results. However, it has been reported that detergents cause the release of certain proteins from the blotting membrane (Flanagan and Yost, 1984). It is therefore advisable to check for this in each experimental system. In our previous paper (Moeremans et al., 1984) we used BSA blocking and noted that colloidal gold probes used without primary antibodies showed non-specific binding to tropomyosin. This binding was inhibited by substituting the dilution buffer of the gold probes with 0.4% gelatin but the use of gelatin is not compatible with the procedure described here. Hence, it is recommended that possible nonspecific binding of gold probes is checked by the omission of the primary antibody. In conclusion, the method presented here extends the potential of blot overlay techniques.
Indeed both the assessment of accuracy of the protein transfer and the comparison of the detected components with the complete protein electropherogram can now be done with high, balanced sensitivity on one blotting membrane. The method is, therefore, a useful and economic modification of potentially wide use.
Acknowledgement We are grateful to Dr. Lydie Meheus (State University off Ghent) for advice and assistance. This work was supported by a grant from the Instituut voor Aanmoediging van Wetenschappelijk Onderzoek in Nijverheid en Landbouw (I.W.O.N.L.), Brussels.
References Batteiger, B., W.J. Newhall V and R.B. Jones, 1982, J. lmmunol. Methods 55, 297. Blattler, D.P., F. Garner, K. Van Slyke and A. Bradley, 1972, J. Chromatogr. 64, 147. Flanagan, S.D. and B. Yost, 1984, Anal. Biochem. 140, 510. Garrels, J.l., 1979, J. Biol. Chem. 254, 1971. Gershoni, J.M. and G.E. Palade, 1983, Anal. Biochem. 131, 1. Hancock, K. and V.C.W. Tsang, 1983, Anal. Biochem. 133, 157. Moeremans, M., G. Daneels, A. Van Dijck, G. Langanger and J~ De Mey, 1984, J. Immunol. Methods 74, 353. Moeremans, M., G. Daneels and J. De Mey, 1985, Anal. Biochem. 145, 315. Moeremans, M., M. De Raeymaeker, G. Daneels and J. De Mey, 1986, Anal. Biochem., 153, 18. Morissey, J.H., 1981, Anal. Biochem. 117, 307. Towbin, H., T. Staehelin and J. Gordon, 1979, Proc. Natl. Acad. Sci. U.S.A. 76, 4350.
89
Elsevier JIM 03888
Sequential immunostaining (gold/silver) and complete protein staining (AuroDye) on Western blots Guy Daneels, Marc Moeremans, Marc De Raeymaeker and Jan De Mey Laboratory of Biochemical Cytology, Division of Cellular Biology and Chemotherapy, Department of Life Sciences. Janssen Pharmaceutica Research Laboratories, B-2340 Beerse, Belgium
(Received 20 November 1985, accepted 11 December 1985)
A double staining method is described which combines immunodetection with sensitive staining of the complete electropherogram on the same membrane. The method is based on the use of Tween 20 as blocking agent, and uses immunogold/silver staining of specific antigens and gold staining of the overall protein pattern with AuroDye. This double staining makes possible the exact location of an immunodetected band within a complex protein pattern. Key words: Colloidal gold," Colloidal iron," lmmunogold/silver staining," Protein blotting," Immunoblotting," Electrophoresis
Introduction
Overlay techniques for electrophoretically separated proteins transferred to immobilizing membranes have become important tools for the analysis of binding activities of different kinds of proteins, especially antibodies (for review see Gershoni and Palade, 1983). In order to make a comparison between an immunodetected band and the total electropherogram one blot or blot unit is usually immunostained and a duplicate stained for the overall protein pattern. Recently we have shown that colloidal gold particles linked to secondary antibodies (Moeremans et al., 1984) and colloidal metal particles (Moeremans et al., 1985, 1986) can be used for sensitive immunostaining and complete protein staining, respectively. Because the preparation of a duplicate blot may sometimes be difficult and is of course both time and sample Abbreviations: PBS-TW, phosphate-buffered saline supplemented with Tween 20; 1D and 2D: one- and two-dimensional gel electrophoresis; NEPHGE: non-equilibrium pH gradient electrophoresis; SDS: sodium dodecyl sulphate.
consuming, especially for 2D blots, we have developed a method that allows for double staining on the same blot. It is based on the observation (Batteiger et al., 1982) that the use of Tween 20 as blocking agent makes it possible to stain the overall protein pattern after specific probing with 125Ilabelled antibodies and before autoradiography. The method presented here has high sensitivity for both immunodetection and complete protein staining. It uses immunogold/silver staining followed by gold staining of the overall protein pattern.
Materials and methods
All reagents were of analytical grade quality and double distilled water was used throughout. A whole cell extract of rat hepatoma cells (HTC) was prepared for 2D electrophoresis according to Garrels (1979). After N E P H G E in the first dimension, proteins were separated according to their molecular weight by SDS electrophoresis (Blattler et al., 1972) and the separated proteins were elec-
0022-1759/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
90 trotransferred to a nitrocellulose membrane (Towbin et al., 1979). Care was taken to place an additional membrane at the cathodic side in the sandwich to adsorb impurities from the blotting system. After transfer, the blot was saturated with PBS (without Ca 2+ and Mg 2+, Gibco), pH 7.2, containing 0.3% Tween 20 (polyoxyethylene sorbitan monolaureate, Bio-Rad), PBS-TW, for 30 min at 37°C and washed 3 x 10 min with PBS-TW at room temperature. All subsequent steps were
!
A
carried out at room temperature. The blot was incubated with a rabbit antibody directed against dog brain tubulin. The antibody was diluted in PBS-0.05% Tween 20 to a concentration of 1 /~g/ml. After incubation for 2 h, the blot was washed (3 x 5 min) with PBS-0.05% TW and the specific interaction was visualized by incubation in goat anti-rabbit globulin linked to 20 nm gold particles (AuroProbe BL GAR, Janssen Life Sciences Products, Beerse, Belgium) for 2 h. This immunogold probe was diluted with PBS-0.05% TW to an optical density at 520 nm of 0.05. After washing in PBS-0.05% TW (3 × 5 min) and in double distilled water (3 × 5 min) this signal was silver-enhanced and fixed (Moeremans et al., 1984) using the IntenSE silver enhancement kit (Janssen Life Sciences Products, Beerse, Belgium). The blot was washed (3 × 5 min) with a large excess of distilled water and a picture was taken. Before processing for gold staining with AuroDye (Janssen Life Sciences Products, Beerse, Belgium) the blot was incubated for 5 rain in PBS 0.3% Tween 20. Then the blot was incubated overnight in the gold stain. After rinsing in excess water the blot was air-dried.
Results In order to demonstrate the utility of this method a whole cell extract of rat hepatoma cells was prepared and run on a 2D gel. After electrotransfer, the nitrocellulose membrane was saturated with Tween 20. The use of Tween 20 as blocking agent did not influence the subsequent immunostaining (Fig. 1A). As shown in Fig. 1B the subsequent protein staining with AuroDye was also not affected by the preceding incubation steps with PBS-TW and the immunogold/silver staining. It showed the high sensitivity as could be expected from this colloidal gold stain.
m
Discussion
Q
B Fig. 1. lmmunodetection (indirect immunogold/silverstaining) of tubulin (arrow) with rabbit anti-tubulin (A). Sequential overall protein staining of the same blot with AuroDye (B).
Using Tween 20 as a blocking agent (Batteiger et al., 1982) one can perform sequential imm u n o a u t o r a d i o g r a p h y and complete protein staining on the same blot. The results presented
91
here show that this can be done with high, matching sensitivity, both detection methods generating stains on the blot. The colloidal iron stain (FerriDye, Janssen Life Sciences Products, Beerse, Belgium) can also be used as a complete protein stain (results not shown). Other overlay techniques (using, for example, lectins) and detection systems can probably be used in the specific probing step. The high contrast provided by the silver enhancement is however a distinct advantage. It is sometimes difficult to distinguish weak immunodetected bands within the complete protein pattern. This problem is easily overcome by taking a picture before complete protein staining. In general the complete protein staining is not only helpful for defining the exact localization of selectively stained components, but also as a quality control of the protein transfer. In our hands, the use of Tween 20, a detergent, did not influence the results. However, it has been reported that detergents cause the release of certain proteins from the blotting membrane (Flanagan and Yost, 1984). It is therefore advisable to check for this in each experimental system. In our previous paper (Moeremans et al., 1984) we used BSA blocking and noted that colloidal gold probes used without primary antibodies showed non-specific binding to tropomyosin. This binding was inhibited by substituting the dilution buffer of the gold probes with 0.4% gelatin but the use of gelatin is not compatible with the procedure described here. Hence, it is recommended that possible nonspecific binding of gold probes is checked by the omission of the primary antibody. In conclusion, the method presented here extends the potential of blot overlay techniques.
Indeed both the assessment of accuracy of the protein transfer and the comparison of the detected components with the complete protein electropherogram can now be done with high, balanced sensitivity on one blotting membrane. The method is, therefore, a useful and economic modification of potentially wide use.
Acknowledgement We are grateful to Dr. Lydie Meheus (State University off Ghent) for advice and assistance. This work was supported by a grant from the Instituut voor Aanmoediging van Wetenschappelijk Onderzoek in Nijverheid en Landbouw (I.W.O.N.L.), Brussels.
References Batteiger, B., W.J. Newhall V and R.B. Jones, 1982, J. lmmunol. Methods 55, 297. Blattler, D.P., F. Garner, K. Van Slyke and A. Bradley, 1972, J. Chromatogr. 64, 147. Flanagan, S.D. and B. Yost, 1984, Anal. Biochem. 140, 510. Garrels, J.l., 1979, J. Biol. Chem. 254, 1971. Gershoni, J.M. and G.E. Palade, 1983, Anal. Biochem. 131, 1. Hancock, K. and V.C.W. Tsang, 1983, Anal. Biochem. 133, 157. Moeremans, M., G. Daneels, A. Van Dijck, G. Langanger and J~ De Mey, 1984, J. Immunol. Methods 74, 353. Moeremans, M., G. Daneels and J. De Mey, 1985, Anal. Biochem. 145, 315. Moeremans, M., M. De Raeymaeker, G. Daneels and J. De Mey, 1986, Anal. Biochem., 153, 18. Morissey, J.H., 1981, Anal. Biochem. 117, 307. Towbin, H., T. Staehelin and J. Gordon, 1979, Proc. Natl. Acad. Sci. U.S.A. 76, 4350.