Electrophoresis 1991, 12, 683-684
Nizar M. Abuharfeil Ragheb F. Atmeh Mahmoud N. Abo-Shehada Saeb N. El-Sukhon Department of Allied Health Sciences, Faculty of Medicine, Jordan University of Science and Technology, Irbid
Fluorescent antibodies in immunoblotting
683
Detection of proteins after immunoblotting on nitrocellulose using fluorescent antibodies A Western blot method is described utilizing 5-dimethylaminonaphthalene- l-sulfonyl (dansyl chloride)-conjugated antibodies to detect proteins transferred onto nitrocellulose. Four proteins of different molecular weight, including human albumin, gamma heavy chain, kappa and lambda light chains of immunoglobulins, were tested. Labeling with the fluorochrome was compared with the method of enzyme-conjugated antibodies and was found to have similar sensitivity, specificity, and safety.Moreover, this method is less expensive, and the labeled antibodies can be prepared fresh in a short time and are free from the disadvantages of enzyme label whose activity is affected by the presence of activators or inhibitors in the tested specimens.
In recent years, immunoblotting of proteins, separated by polyacrylamide gel electrophoresis and transferred onto nitrocellulose (NC), has become a valuable and popular technique [ 1,2].The antibodies are labeled with an enzyme that most commonly used alkaline phosphatase or horseradish peroxidase (HRP). The primary criteria for selection of the conjugated enzyme are that the enzyme should be stable under the conditions used for storage, cross-linking and assay, should have a high specific activity and be inexpensive. Equally important is that the enzyme may not be present in the antigen preparation to be used in the serologic tests; otherwise, false positive tests would result. On the other hand, false negative results could be obtained from the presence of enzyme inhibitors or inactivators in serologic reagents such as sodium azide, which is commonly used as a preservative and has an inhibitory effect on the peroxidase label [3]. Conjugation with alkaline phosphatase, due to its relatively high molecular weight (MI 100000), results in conjugates with low activity [4].The use of fluorescent dyes for the detection of transferred proteins on NC is an interesting but so far neglected alternative [l]. In this communication the use of a simple, inexpensive alternative label, the fluorochrome 5-dimethylaminonaphthalene-1-sulfonyl chloride (dansyl chloride) avoiding the disadvantages of the enzyme label, is presented. Blood was collected from healthy donors and serum was separated by low-speed centrifugation. Anti-human albumin, anti-kappa light chain, anti-lambda light chain, and anti-gamma heavy chain were obtained from Pharmacia (Uppsala, Sweden). Goat anti-rabbit IgG and goat anti-rabbit IgG-horseradish peroxidase (HRP) conjugate were purchased from Bio-Rad (Richmond, CA). Human albumin and 1-dimethylamino-naphthalene-5-sulphonyl(dansyl) chloride were obtained from Sigma (St. Louis, MO). All chemical reagents were of analytical grade. Diluted normal human serum (NHS) or human albumin samples were run on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) according to [5], using 5-20°/o gradient polyacrylamide gels. The samples were transferred onto NC sheets using the Bio-Rad Transblot apparatus, employing as buffer 15.6 mM Tris, 120 mM glycine, pH 8.6,conCorrespondence: Dr. N. Abuharfeil, Department of Biological Sciences, Faculty of Science, Jordan University of Science and Technology, Irbid, Jordan Abbreviation: dansyl, 5-dimethylaminonaphthalene-1-sulfonyl;HRP, horseradish peroxidase; NC,nitrocellulose; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TBS,Tris buffered saline; TTBS, Tween-20 in TBS
0VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1991
taining 20% methanol and 0.03 O/o SDS. Antibody dansylation was done by a modification of the method of Talbot and Yphantis [61. In brief, the protein sample (25 mg) was dissolved in 1 mL of 0.05 M Na,HPO,, pH 8.2. A 10% solution of dansyl chloride in acetone was added, at a ratio of20 pL per one mL of protein solution, with vigorous shaking. The low molecular weight forms of the dye were removed by gel filtration on Sephadex G-25 (Pharmacia) using a small column (7 mL gel). The absorbance of the collected fractions was measured spectrophotometrically at 280 nm and used for the protein estimation and their fluorescence was examined fluorometrically in a dark room at 366 nm. Fractions with a high protein content and high fluorescence were collected and diluted to 150 mL with 20 mM Tris, 500 mM NaC1, pH 7.5 (TBS), containing 0.05% Tween 20 (TTBS). Antibody activity was checked before and after dansylation by using the single radial immunodiffusion technique [7]. Four antibodies, i. e. anti-human albumin, anti-gamma heavy chain, anti-kappa light chain and antilambda light chain of immunoglobulin, were conjugated separately. For immunoblotting with dansyl label, NC sheets were treated at room temperature as follows: (1) washed in 3 % gelatin in TBS for 2 X 5 min (blocking step); (2) incubated with the first antibody (rabbit antiserum) for 2 h or overnight; (3) washed 2 X 5 min with TTBS; (4) incubated with the second antibody (goat anti-rabbit IgG-dansyl conjugate for 2,4,6,8,10,16 h; ( 5 ) washed 2 X 5 min with TTBS; (6) washed with distilled water for 10 min. Results were examined in a dark room illuminated with a longwave UV lamp (366 nm). For immunoblotting with enzyme label, the steps mentioned in the dansyl label were repeated, except step (4), which was performed by the incubation with the second antibody (anti rabbit IgG-HRP) for 1h. After the blot had been washed with TBS for 2 X 5 min, it was immersed in development solution (4-chloro-1-naphthol, 0.06% w/v in methanol plus 0.01 O/o H,O,) for30 min; (8) development was stopped by immersing in distilled water for 10 min. The results of immunoblots obtained by analysis of different quantities of human serum albumin using anti-rabbit IgG-HRP conjugate and anti-rabbit IgG-dansyl chloride conjugate are shown in Fig. 1. No difference in sensitivity could be observed between the two methods. The least amount detected was 20 ng of albumin. For optimal conditions, the incubation with dansylated antibody was tested at different intervals. It was found that optimal results can 0173-0835/91/0909-0683 $3.50+.25/0
684
Electrophoresis 1991, 12,683-684
N. M. Abuharfeil er a /
Figure 1. (A) Western blot of human serum albumin. After SDS-PAGE (5-20% gradient acrylamide gel) and transfer to NC membrane, the protein samples were immunoprecipitated with rabbit anti-human albumin followed by (A) anti-rabbit IgG-dansyl-conjugated antibodies or (B) antirabbit IgG-HRP conjugated antibodies. Lanes (1)-(4) contain 0.4 pg, 0.2 pg, 0.1 pg, and 20 ng of human serum albumin, respectively.
be obtained after 8 h. Similar results were obtained when different quantities of normal human serum (2,3,4, and 5 pL) at 1:2000 dilution were analyzed with Western blot using anti-human albumin conjugated with dansyl or HRP. One protein band was obtained and the least amount to be detected was 2 pL. Moreover, the results of the Mancini test demonstrated that the antibody activity was not affected due to the dansylation process (data not shown). In order to test the possible application of the method to other proteins, lambda light chain, kappa light chain and gamma heavy chain of Ig were tested in normal human serum (2 pL at 1:25,1:125, and 1:400 dilution, respectively) (Fig. 2). No difference could be demonstrated regarding the sensitivity and specificity of the antibodies by using either the enzyme or the fluorochrome conjugate. Four different proteins were used for the comparison between the fluorescent and enzyme 1abels.The results showed that the use of fluorescent label described above for Western blots maintain the advantages of the enzyme label, including the sensitivity (Fig. l),specificity, and safety, especially in those cases where the enzyme could not be used. Moreover, this method is less expensive and includes less steps than the enzyme label, which also requires a visualization step and the use of a substrate. The labeled antibody is stable and can be preserved by the use of sodium azide, which is an enzyme inhibitor. It can be applied in those cases where the enzyme could not be used due to the presence of the enzyme itself or its activators or inhibitors in the samples.The method could be used for semiquantitation or detection of different immunoreactive proteins in serum after electrophoretic transfer and immunoblotting onto NC. Quantitation of different protein bands on the same lane cannot be done by Western blot, using either the enzyme or fluorochrome labels, as electrotransfer is generally dependent on the molecular weight of individual proteins [S]. The antibody activity did not change upon dansylation; however, it is important to separate the low molecular
Figure 2. Western blot of normal human serum proteins. After SDSPAGE (5-20°/0 gradient acrylamide), and NC transfer, normal human Serum proteins were immunoprecipitated with rabbit anti-human (1) kappa light chain o f k , (2) lambda light chain ofIg (3) gamma heavy chain of Ig followed by (A) anti-rabbit IgG-dansyl conjugated antibodies or (B) anti-rabbit IgG HRP conjugated antibodies.
weight fluorochrome efficiently, otherwise nonspecific bands might appear. The color of the enzyme-labeled Western blots decreases relatively quickly, especially for HRP, and also the background changes upon prolonged storage. The intensity of fluorochrome bands decreases much slower than that of the enzyme-labeled bands, and also the background contrast is sharper. The fluorochrome blots could be preserved for two months without significant loss of fluorescence and without the background being changed. Also, it is much easier to scan the fluorochrome protein bands than the colored enzyme-labeled bands due to the high background color in the latter, particularly when using HRP. However, in order to obtain equal sensitivity in the two methods, the blots should be incubated with the dansylated antibody for a longer time than with the enzyme-antibody conjugate. This increase in time is well compensated by fewer steps in the dansylation method. The presented method affords advantages over the enzyme-label procedure and may prove a useful alternative for labeling Western blots. This work was supported by the Jordan Universiv of Science and Technology, Research Project 21/88. Received December 21, 1991
References [l] Towbin, H., Staehelin, T. and Gordon, J., Proc. Natl. Acad. Sci. USA 1979, 76, 4350-4354. [2] Towbin, H. and Gordon, J. J. Immunol. Methods 1984, 72,313-340. [ 3 ] Avrameas, S . , Curr. Top. Microbiol. Zmmunol. 1983, 104,93-103. [4] Yolken, R. H., Rev. Infect. Diseases 1982, 4,35-68. [S] Laemmli, U. K., Nature 1970,227, 680-685. [6] Talbot, D. N. and Yphantis, D. A,, Anal. Biochem. 1965,44,246-253. [7] Mancini,G.,Carbonara,A. 0.andHeremans,J.F.,Immunochemistry 1965,2,235-254. [8] Gershoni, J. M. and Palade, G. E., Anal. Biochem. 1983, 131, 1-15.
Nizar M. Abuharfeil Ragheb F. Atmeh Mahmoud N. Abo-Shehada Saeb N. El-Sukhon Department of Allied Health Sciences, Faculty of Medicine, Jordan University of Science and Technology, Irbid
Fluorescent antibodies in immunoblotting
683
Detection of proteins after immunoblotting on nitrocellulose using fluorescent antibodies A Western blot method is described utilizing 5-dimethylaminonaphthalene- l-sulfonyl (dansyl chloride)-conjugated antibodies to detect proteins transferred onto nitrocellulose. Four proteins of different molecular weight, including human albumin, gamma heavy chain, kappa and lambda light chains of immunoglobulins, were tested. Labeling with the fluorochrome was compared with the method of enzyme-conjugated antibodies and was found to have similar sensitivity, specificity, and safety.Moreover, this method is less expensive, and the labeled antibodies can be prepared fresh in a short time and are free from the disadvantages of enzyme label whose activity is affected by the presence of activators or inhibitors in the tested specimens.
In recent years, immunoblotting of proteins, separated by polyacrylamide gel electrophoresis and transferred onto nitrocellulose (NC), has become a valuable and popular technique [ 1,2].The antibodies are labeled with an enzyme that most commonly used alkaline phosphatase or horseradish peroxidase (HRP). The primary criteria for selection of the conjugated enzyme are that the enzyme should be stable under the conditions used for storage, cross-linking and assay, should have a high specific activity and be inexpensive. Equally important is that the enzyme may not be present in the antigen preparation to be used in the serologic tests; otherwise, false positive tests would result. On the other hand, false negative results could be obtained from the presence of enzyme inhibitors or inactivators in serologic reagents such as sodium azide, which is commonly used as a preservative and has an inhibitory effect on the peroxidase label [3]. Conjugation with alkaline phosphatase, due to its relatively high molecular weight (MI 100000), results in conjugates with low activity [4].The use of fluorescent dyes for the detection of transferred proteins on NC is an interesting but so far neglected alternative [l]. In this communication the use of a simple, inexpensive alternative label, the fluorochrome 5-dimethylaminonaphthalene-1-sulfonyl chloride (dansyl chloride) avoiding the disadvantages of the enzyme label, is presented. Blood was collected from healthy donors and serum was separated by low-speed centrifugation. Anti-human albumin, anti-kappa light chain, anti-lambda light chain, and anti-gamma heavy chain were obtained from Pharmacia (Uppsala, Sweden). Goat anti-rabbit IgG and goat anti-rabbit IgG-horseradish peroxidase (HRP) conjugate were purchased from Bio-Rad (Richmond, CA). Human albumin and 1-dimethylamino-naphthalene-5-sulphonyl(dansyl) chloride were obtained from Sigma (St. Louis, MO). All chemical reagents were of analytical grade. Diluted normal human serum (NHS) or human albumin samples were run on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) according to [5], using 5-20°/o gradient polyacrylamide gels. The samples were transferred onto NC sheets using the Bio-Rad Transblot apparatus, employing as buffer 15.6 mM Tris, 120 mM glycine, pH 8.6,conCorrespondence: Dr. N. Abuharfeil, Department of Biological Sciences, Faculty of Science, Jordan University of Science and Technology, Irbid, Jordan Abbreviation: dansyl, 5-dimethylaminonaphthalene-1-sulfonyl;HRP, horseradish peroxidase; NC,nitrocellulose; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TBS,Tris buffered saline; TTBS, Tween-20 in TBS
0VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1991
taining 20% methanol and 0.03 O/o SDS. Antibody dansylation was done by a modification of the method of Talbot and Yphantis [61. In brief, the protein sample (25 mg) was dissolved in 1 mL of 0.05 M Na,HPO,, pH 8.2. A 10% solution of dansyl chloride in acetone was added, at a ratio of20 pL per one mL of protein solution, with vigorous shaking. The low molecular weight forms of the dye were removed by gel filtration on Sephadex G-25 (Pharmacia) using a small column (7 mL gel). The absorbance of the collected fractions was measured spectrophotometrically at 280 nm and used for the protein estimation and their fluorescence was examined fluorometrically in a dark room at 366 nm. Fractions with a high protein content and high fluorescence were collected and diluted to 150 mL with 20 mM Tris, 500 mM NaC1, pH 7.5 (TBS), containing 0.05% Tween 20 (TTBS). Antibody activity was checked before and after dansylation by using the single radial immunodiffusion technique [7]. Four antibodies, i. e. anti-human albumin, anti-gamma heavy chain, anti-kappa light chain and antilambda light chain of immunoglobulin, were conjugated separately. For immunoblotting with dansyl label, NC sheets were treated at room temperature as follows: (1) washed in 3 % gelatin in TBS for 2 X 5 min (blocking step); (2) incubated with the first antibody (rabbit antiserum) for 2 h or overnight; (3) washed 2 X 5 min with TTBS; (4) incubated with the second antibody (goat anti-rabbit IgG-dansyl conjugate for 2,4,6,8,10,16 h; ( 5 ) washed 2 X 5 min with TTBS; (6) washed with distilled water for 10 min. Results were examined in a dark room illuminated with a longwave UV lamp (366 nm). For immunoblotting with enzyme label, the steps mentioned in the dansyl label were repeated, except step (4), which was performed by the incubation with the second antibody (anti rabbit IgG-HRP) for 1h. After the blot had been washed with TBS for 2 X 5 min, it was immersed in development solution (4-chloro-1-naphthol, 0.06% w/v in methanol plus 0.01 O/o H,O,) for30 min; (8) development was stopped by immersing in distilled water for 10 min. The results of immunoblots obtained by analysis of different quantities of human serum albumin using anti-rabbit IgG-HRP conjugate and anti-rabbit IgG-dansyl chloride conjugate are shown in Fig. 1. No difference in sensitivity could be observed between the two methods. The least amount detected was 20 ng of albumin. For optimal conditions, the incubation with dansylated antibody was tested at different intervals. It was found that optimal results can 0173-0835/91/0909-0683 $3.50+.25/0
684
Electrophoresis 1991, 12,683-684
N. M. Abuharfeil er a /
Figure 1. (A) Western blot of human serum albumin. After SDS-PAGE (5-20% gradient acrylamide gel) and transfer to NC membrane, the protein samples were immunoprecipitated with rabbit anti-human albumin followed by (A) anti-rabbit IgG-dansyl-conjugated antibodies or (B) antirabbit IgG-HRP conjugated antibodies. Lanes (1)-(4) contain 0.4 pg, 0.2 pg, 0.1 pg, and 20 ng of human serum albumin, respectively.
be obtained after 8 h. Similar results were obtained when different quantities of normal human serum (2,3,4, and 5 pL) at 1:2000 dilution were analyzed with Western blot using anti-human albumin conjugated with dansyl or HRP. One protein band was obtained and the least amount to be detected was 2 pL. Moreover, the results of the Mancini test demonstrated that the antibody activity was not affected due to the dansylation process (data not shown). In order to test the possible application of the method to other proteins, lambda light chain, kappa light chain and gamma heavy chain of Ig were tested in normal human serum (2 pL at 1:25,1:125, and 1:400 dilution, respectively) (Fig. 2). No difference could be demonstrated regarding the sensitivity and specificity of the antibodies by using either the enzyme or the fluorochrome conjugate. Four different proteins were used for the comparison between the fluorescent and enzyme 1abels.The results showed that the use of fluorescent label described above for Western blots maintain the advantages of the enzyme label, including the sensitivity (Fig. l),specificity, and safety, especially in those cases where the enzyme could not be used. Moreover, this method is less expensive and includes less steps than the enzyme label, which also requires a visualization step and the use of a substrate. The labeled antibody is stable and can be preserved by the use of sodium azide, which is an enzyme inhibitor. It can be applied in those cases where the enzyme could not be used due to the presence of the enzyme itself or its activators or inhibitors in the samples.The method could be used for semiquantitation or detection of different immunoreactive proteins in serum after electrophoretic transfer and immunoblotting onto NC. Quantitation of different protein bands on the same lane cannot be done by Western blot, using either the enzyme or fluorochrome labels, as electrotransfer is generally dependent on the molecular weight of individual proteins [S]. The antibody activity did not change upon dansylation; however, it is important to separate the low molecular
Figure 2. Western blot of normal human serum proteins. After SDSPAGE (5-20°/0 gradient acrylamide), and NC transfer, normal human Serum proteins were immunoprecipitated with rabbit anti-human (1) kappa light chain o f k , (2) lambda light chain ofIg (3) gamma heavy chain of Ig followed by (A) anti-rabbit IgG-dansyl conjugated antibodies or (B) anti-rabbit IgG HRP conjugated antibodies.
weight fluorochrome efficiently, otherwise nonspecific bands might appear. The color of the enzyme-labeled Western blots decreases relatively quickly, especially for HRP, and also the background changes upon prolonged storage. The intensity of fluorochrome bands decreases much slower than that of the enzyme-labeled bands, and also the background contrast is sharper. The fluorochrome blots could be preserved for two months without significant loss of fluorescence and without the background being changed. Also, it is much easier to scan the fluorochrome protein bands than the colored enzyme-labeled bands due to the high background color in the latter, particularly when using HRP. However, in order to obtain equal sensitivity in the two methods, the blots should be incubated with the dansylated antibody for a longer time than with the enzyme-antibody conjugate. This increase in time is well compensated by fewer steps in the dansylation method. The presented method affords advantages over the enzyme-label procedure and may prove a useful alternative for labeling Western blots. This work was supported by the Jordan Universiv of Science and Technology, Research Project 21/88. Received December 21, 1991
References [l] Towbin, H., Staehelin, T. and Gordon, J., Proc. Natl. Acad. Sci. USA 1979, 76, 4350-4354. [2] Towbin, H. and Gordon, J. J. Immunol. Methods 1984, 72,313-340. [ 3 ] Avrameas, S . , Curr. Top. Microbiol. Zmmunol. 1983, 104,93-103. [4] Yolken, R. H., Rev. Infect. Diseases 1982, 4,35-68. [S] Laemmli, U. K., Nature 1970,227, 680-685. [6] Talbot, D. N. and Yphantis, D. A,, Anal. Biochem. 1965,44,246-253. [7] Mancini,G.,Carbonara,A. 0.andHeremans,J.F.,Immunochemistry 1965,2,235-254. [8] Gershoni, J. M. and Palade, G. E., Anal. Biochem. 1983, 131, 1-15.
