- Email: [email protected]
An Apparatus for Immunoblotting Using a Magnetic Rubber Sheet
230
NOTES & TIPS
nant polypeptide may be an indispensable step toward any study supposed to reflect the physiological state. Acknowledgments. We thank Dr. T. Rein for valuable comments and discussions and Dr. E.-L. Winnacker for continuous support. This work was supported by a grant from the Deutsche Forschungsgemeinschaft to H.Z. (DFG Zo 59/2-1).
REFERENCES 1. Crowe, J., and Henco, K. (1992) The Qiaexpressionist, 2nd ed., Diagen GmbH, Hilden. 2. Kieran, M., Blank, V., Logeat, F., Vandekerchove, J., Lottspeich, F., LeBail, O., Urban, M. B., Kourilsky, P., Baeuerle, P. A., and Israel, A. (1990) Cell 62, 1007–1018. 3. Sen, R., and Baltimore, D. (1986) Cell 47, 921–928. 4. Bu¨ning, H., Baeuerle, P. A., and Zorbas, H. (1995) Nucleic Acids Res. 23, 1443–1444. 5. Clark, L., Nicholson, J., and Hay, R. T. (1989) J. Mol. Biol. 206, 615–626. 6. Gosh, G., Duyne, G. v., Gosh, s., and Sigler, P. B. (1995) Nature 373, 303–310. 7. Mu¨ller, C. W., Rey, F. A., Sodeoka, M., Verdine, G. L., and Harrison, S. C. (1995) Nature 373, 311–317.
An Apparatus for Immunoblotting Using a Magnetic Rubber Sheet Atsushi Tabata and Masahiro Uritani1 Department of Chemistry, Faculty of Science, Shizuoka University, 836 Oya, Shizuoka 422, Japan Received October 16, 1995
Immunoblotting combines the resolution of gel electrophoresis with the specificity of immunochemical detection (1). It can be used to determine the presence, quantity, and the relative molecular weight of a protein antigen. Immunoblotting is also a particularly powerful technique for assaying the presence, quantity, and specificity of antibodies from different samples of polyclonal sera. To determine the characteristics of different antibodies at the same time, a membrane onto which proteins are transferred is usually cut into strips, and each strip is immunostained with each antibody sample. Alternatively, the membrane, without cutting, is set in a special apparatus that partitions the membrane into small narrow pieces, and each piece is treated with each antibody at the same time. Such an apparatus is commercially available (for example, Mini-PROTEAN II multiscreen apparatus from BioRad), however, it is very expensive. In this paper, we 1
To whom correspondence should be addressed. Fax: (054) 2373384. ANALYTICAL BIOCHEMISTRY ARTICLE NO.
describe the preparation and use of an apparatus using a magnetic rubber sheet. This apparatus is easily and economically prepared, and it works well. Materials and Methods Preparation of the apparatus. A magnetic rubber sheet (20 cm 1 10 cm 1 0.8 mm) was obtained from Niigata Seiki Co., Ltd. (Niigata, Japan), which was available in do-it-yourself shops. Two equal sheets were cut to the same size, which were larger than that of the membrane (the size of 10 1 8 cm was good for an ordinary size minislab gel). One of the sheets that was not further processed was referred to as a supporting sheet (Fig. 1). The other sheet was cut with a razor blade to produce slots and was referred to as a multislot sheet. The apparatus consisted of only these two sheets. The width of each slot was slightly narrower than that of each lane on the membrane onto which proteins were blotted. The length of each slot was slightly shorter than that of the membrane. SDS–PAGE 2 and electrotransfer of proteins. The postribosomal supernatant fraction was prepared from yeast as previously described (2). The proteins of the fraction were subjected to SDS–PAGE as previously described (3) using an ordinary apparatus for minislab gel electrophoresis (Nihon Eido, Tokyo, Japan). To detect the position of a lane, prestained protein markers (Amersham) were loaded onto one of the wells. After gel electrophoresis, the proteins were electrotransferred from the gel to a nitrocellulose membrane (0.2 mm, Schleicher & Schuell) in a buffer (50 mM Tris, 380 mM glycine, 0.1% [w/v] SDS, 20% [v/v] methanol) for 2 h at 30 V using a Bio-Rad Mini-Transblot cell. Immunostaining of the proteins. After electrotransfer, the membrane was incubated in TBS (1) with 5% (w/v) nonfat dry milk at 47C overnight. The membrane was carefully put on the multislot sheet so that each lane of the membrane should align with each slot of the multislot sheet and the protein side of the membrane should face the noncoated side of the multislot sheet (the magnetic rubber sheet had been coated with plastic on one side by the manufacturer). The prestained protein markers were good landmarks for the position of the lane. The multislot sheet was then marked at the corners of the membrane. The supporting sheet was then placed on the membrane (Fig. 2A). The two sheets remained together by magnetic attraction, holding the membrane tightly. The noncoated side of the supporting sheet faced the noncoated side of the multislot sheet because the noncoated sides provided a stronger magnetic attraction than the coated sides. 2 Abbreviations used: BCIP, bromochloroindoyl phosphate; NBT, nitro blue tetrazolium; PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulfate; EF, translation elongation factor; TBS, Tris-buffered saline.
234, 230–232 (1996)
0079
0003-2697/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
/ 6m0f$$$nat
01-30-96 01:09:34
abnt
AP-Anal Bio
NOTES & TIPS
231
FIG. 1. The apparatus for immunoblotting using a magnetic rubber sheet.
FIG. 2. Setting of a nitrocellulose membrane in the apparatus (A) and immunostaining of the proteins on the membrane that is set in the apparatus (B).
/ 6m0f$$$nat
01-30-96 01:09:34
abnt
AP-Anal Bio
232
NOTES & TIPS
For immunostaining, antibody solutions (150 ml per lane) that had been diluted 1000-fold with TBS were placed on the membrane that was set in the apparatus (Fig. 2B). After incubation for 1 h at room temperature, the antibody solutions were thoroughly removed with blotting paper, and then the membrane was removed from the apparatus and washed four times with TBS. The membrane was set again in the apparatus (the marks on the multislot sheet showed the correct position of the membrane) and a second-antibody (alkaline phosphatase-conjugated goat anti-rabbit IgG, TAGO) solution (150 ml per lane) that had been diluted 1000fold with TBS was placed on the membrane. After incubation for 1 h at room temperature, the second-antibody solution was thoroughly removed, and then the membrane was removed from the apparatus and washed four times with TBS. The membrane was set again in the apparatus and a substrate solution (150 ml per lane) containing 0.3 mg/ml BCIP, 0.6 mg/ml NBT, 1 mM magnesium chloride, and 0.1 M sodium carbonate buffer (pH 9.8) was placed on the membrane to visualize the antibody. After staining, the membrane was removed from the apparatus and then washed with 0.1 mM EDTA and distilled water. Results and Discussion In this report, we prepared an apparatus for immunoblotting using a magnetic rubber sheet. It was very simple, consisting of only two parts: the supporting sheet and the multislot sheet (Fig. 1). Because the magnetic rubber sheet was soft, it was cut easily with a razor blade, and thus the apparatus was quickly made within an hour. The magnetic rubber sheet cost only a few dollars, so the apparatus was inexpensive. The magnetic rubber sheet provided a magnetic force in all the area, so that the apparatus held the membrane tightly enough to prevent the antibody solution from leaking as will be described below. The results for the immunoblotting using the apparatus are shown in Fig. 3A. The proteins in lanes 1, 2, and 3 were immunostained with different antibodies. There was no obvious common band between lanes 1 and 2, and lanes 2 and 3, which indicates that the antibody solution did not leak and contaminate the antibody solution in the neighboring lanes. The area that had been shielded by the apparatus showed a very low background, indicating that the antibody and the substrate solutions did not leak and stain the membrane. For control, we cut the membrane into strips and immunostained, without using the apparatus, each strip with each antibody (Fig. 3B). The results were the same as those obtained using the apparatus (Fig. 3A). It is important to make the apparatus so that its slots fit the lanes of the membrane but not of the gel because the gel may expand slightly during electro-
/ 6m0f$$$nat
01-30-96 01:09:34
abnt
FIG. 3. Immunoblotting of the yeast postribosomal supernatant fraction with various antibodies using (A) or not using (B) the apparatus. In A and B, the proteins in the postribosomal supernatant fraction (10 mg per lane) were electrophoresed in an SDS–polyacrylamide gel (10%), electrotransferred onto a nitrocellulose membrane, and immunostained with anti-yeast EF-1a antibody (lane 1), anti-yeast EF-2 antibody (lane 2), or anti-yeast EF-3 antibody (lane 3). Lane M, molecular weights of protein markers.
transfer. For such purpose, prestained markers are useful for detecting the position of each lane on the membrane. If a sample is analyzed by SDS–PAGE in a large preparative well, there is no need to make each slot fit each lane. In this case, it is possible to make the multislot sheet with the desired number and widths of the slots. After use, the apparatus should be washed with detergent, rinsed with distilled water, and air dried at room temperature. We used the same apparatus over 20 times and still obtained good results. In summary, we reported the preparation and use of an apparatus for immunoblotting using a magnetic rubber sheet. This apparatus held a membrane tightly enough to prevent leaking of the antibody solution to neighboring lanes although it was prepared and used very easily. Acknowledgments. We thank Prof. Miyazaki (Nagoya University, Japan) for kindly giving us the anti-EF-2 and anti-EF-3 antibodies, and Prof. Ishikawa and Prof. Yoshinaga (Shizuoka University, Japan) for critical reading of the manuscript.
REFERENCES 1. Harlow, E., and Lane, D. (1988) in Antibodies: A Laboratory Manual, pp. 471–510, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. 2. Miyazaki, M., Uritani, M., Fujimura, K., Yamakatsu, H., Kageyama, T., and Takahashi, K. (1988) J. Biochem. 103, 508–521. 3. Laemmli, U. K. (1970) Nature 227, 680–685.
AP-Anal Bio
NOTES & TIPS
nant polypeptide may be an indispensable step toward any study supposed to reflect the physiological state. Acknowledgments. We thank Dr. T. Rein for valuable comments and discussions and Dr. E.-L. Winnacker for continuous support. This work was supported by a grant from the Deutsche Forschungsgemeinschaft to H.Z. (DFG Zo 59/2-1).
REFERENCES 1. Crowe, J., and Henco, K. (1992) The Qiaexpressionist, 2nd ed., Diagen GmbH, Hilden. 2. Kieran, M., Blank, V., Logeat, F., Vandekerchove, J., Lottspeich, F., LeBail, O., Urban, M. B., Kourilsky, P., Baeuerle, P. A., and Israel, A. (1990) Cell 62, 1007–1018. 3. Sen, R., and Baltimore, D. (1986) Cell 47, 921–928. 4. Bu¨ning, H., Baeuerle, P. A., and Zorbas, H. (1995) Nucleic Acids Res. 23, 1443–1444. 5. Clark, L., Nicholson, J., and Hay, R. T. (1989) J. Mol. Biol. 206, 615–626. 6. Gosh, G., Duyne, G. v., Gosh, s., and Sigler, P. B. (1995) Nature 373, 303–310. 7. Mu¨ller, C. W., Rey, F. A., Sodeoka, M., Verdine, G. L., and Harrison, S. C. (1995) Nature 373, 311–317.
An Apparatus for Immunoblotting Using a Magnetic Rubber Sheet Atsushi Tabata and Masahiro Uritani1 Department of Chemistry, Faculty of Science, Shizuoka University, 836 Oya, Shizuoka 422, Japan Received October 16, 1995
Immunoblotting combines the resolution of gel electrophoresis with the specificity of immunochemical detection (1). It can be used to determine the presence, quantity, and the relative molecular weight of a protein antigen. Immunoblotting is also a particularly powerful technique for assaying the presence, quantity, and specificity of antibodies from different samples of polyclonal sera. To determine the characteristics of different antibodies at the same time, a membrane onto which proteins are transferred is usually cut into strips, and each strip is immunostained with each antibody sample. Alternatively, the membrane, without cutting, is set in a special apparatus that partitions the membrane into small narrow pieces, and each piece is treated with each antibody at the same time. Such an apparatus is commercially available (for example, Mini-PROTEAN II multiscreen apparatus from BioRad), however, it is very expensive. In this paper, we 1
To whom correspondence should be addressed. Fax: (054) 2373384. ANALYTICAL BIOCHEMISTRY ARTICLE NO.
describe the preparation and use of an apparatus using a magnetic rubber sheet. This apparatus is easily and economically prepared, and it works well. Materials and Methods Preparation of the apparatus. A magnetic rubber sheet (20 cm 1 10 cm 1 0.8 mm) was obtained from Niigata Seiki Co., Ltd. (Niigata, Japan), which was available in do-it-yourself shops. Two equal sheets were cut to the same size, which were larger than that of the membrane (the size of 10 1 8 cm was good for an ordinary size minislab gel). One of the sheets that was not further processed was referred to as a supporting sheet (Fig. 1). The other sheet was cut with a razor blade to produce slots and was referred to as a multislot sheet. The apparatus consisted of only these two sheets. The width of each slot was slightly narrower than that of each lane on the membrane onto which proteins were blotted. The length of each slot was slightly shorter than that of the membrane. SDS–PAGE 2 and electrotransfer of proteins. The postribosomal supernatant fraction was prepared from yeast as previously described (2). The proteins of the fraction were subjected to SDS–PAGE as previously described (3) using an ordinary apparatus for minislab gel electrophoresis (Nihon Eido, Tokyo, Japan). To detect the position of a lane, prestained protein markers (Amersham) were loaded onto one of the wells. After gel electrophoresis, the proteins were electrotransferred from the gel to a nitrocellulose membrane (0.2 mm, Schleicher & Schuell) in a buffer (50 mM Tris, 380 mM glycine, 0.1% [w/v] SDS, 20% [v/v] methanol) for 2 h at 30 V using a Bio-Rad Mini-Transblot cell. Immunostaining of the proteins. After electrotransfer, the membrane was incubated in TBS (1) with 5% (w/v) nonfat dry milk at 47C overnight. The membrane was carefully put on the multislot sheet so that each lane of the membrane should align with each slot of the multislot sheet and the protein side of the membrane should face the noncoated side of the multislot sheet (the magnetic rubber sheet had been coated with plastic on one side by the manufacturer). The prestained protein markers were good landmarks for the position of the lane. The multislot sheet was then marked at the corners of the membrane. The supporting sheet was then placed on the membrane (Fig. 2A). The two sheets remained together by magnetic attraction, holding the membrane tightly. The noncoated side of the supporting sheet faced the noncoated side of the multislot sheet because the noncoated sides provided a stronger magnetic attraction than the coated sides. 2 Abbreviations used: BCIP, bromochloroindoyl phosphate; NBT, nitro blue tetrazolium; PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulfate; EF, translation elongation factor; TBS, Tris-buffered saline.
234, 230–232 (1996)
0079
0003-2697/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
/ 6m0f$$$nat
01-30-96 01:09:34
abnt
AP-Anal Bio
NOTES & TIPS
231
FIG. 1. The apparatus for immunoblotting using a magnetic rubber sheet.
FIG. 2. Setting of a nitrocellulose membrane in the apparatus (A) and immunostaining of the proteins on the membrane that is set in the apparatus (B).
/ 6m0f$$$nat
01-30-96 01:09:34
abnt
AP-Anal Bio
232
NOTES & TIPS
For immunostaining, antibody solutions (150 ml per lane) that had been diluted 1000-fold with TBS were placed on the membrane that was set in the apparatus (Fig. 2B). After incubation for 1 h at room temperature, the antibody solutions were thoroughly removed with blotting paper, and then the membrane was removed from the apparatus and washed four times with TBS. The membrane was set again in the apparatus (the marks on the multislot sheet showed the correct position of the membrane) and a second-antibody (alkaline phosphatase-conjugated goat anti-rabbit IgG, TAGO) solution (150 ml per lane) that had been diluted 1000fold with TBS was placed on the membrane. After incubation for 1 h at room temperature, the second-antibody solution was thoroughly removed, and then the membrane was removed from the apparatus and washed four times with TBS. The membrane was set again in the apparatus and a substrate solution (150 ml per lane) containing 0.3 mg/ml BCIP, 0.6 mg/ml NBT, 1 mM magnesium chloride, and 0.1 M sodium carbonate buffer (pH 9.8) was placed on the membrane to visualize the antibody. After staining, the membrane was removed from the apparatus and then washed with 0.1 mM EDTA and distilled water. Results and Discussion In this report, we prepared an apparatus for immunoblotting using a magnetic rubber sheet. It was very simple, consisting of only two parts: the supporting sheet and the multislot sheet (Fig. 1). Because the magnetic rubber sheet was soft, it was cut easily with a razor blade, and thus the apparatus was quickly made within an hour. The magnetic rubber sheet cost only a few dollars, so the apparatus was inexpensive. The magnetic rubber sheet provided a magnetic force in all the area, so that the apparatus held the membrane tightly enough to prevent the antibody solution from leaking as will be described below. The results for the immunoblotting using the apparatus are shown in Fig. 3A. The proteins in lanes 1, 2, and 3 were immunostained with different antibodies. There was no obvious common band between lanes 1 and 2, and lanes 2 and 3, which indicates that the antibody solution did not leak and contaminate the antibody solution in the neighboring lanes. The area that had been shielded by the apparatus showed a very low background, indicating that the antibody and the substrate solutions did not leak and stain the membrane. For control, we cut the membrane into strips and immunostained, without using the apparatus, each strip with each antibody (Fig. 3B). The results were the same as those obtained using the apparatus (Fig. 3A). It is important to make the apparatus so that its slots fit the lanes of the membrane but not of the gel because the gel may expand slightly during electro-
/ 6m0f$$$nat
01-30-96 01:09:34
abnt
FIG. 3. Immunoblotting of the yeast postribosomal supernatant fraction with various antibodies using (A) or not using (B) the apparatus. In A and B, the proteins in the postribosomal supernatant fraction (10 mg per lane) were electrophoresed in an SDS–polyacrylamide gel (10%), electrotransferred onto a nitrocellulose membrane, and immunostained with anti-yeast EF-1a antibody (lane 1), anti-yeast EF-2 antibody (lane 2), or anti-yeast EF-3 antibody (lane 3). Lane M, molecular weights of protein markers.
transfer. For such purpose, prestained markers are useful for detecting the position of each lane on the membrane. If a sample is analyzed by SDS–PAGE in a large preparative well, there is no need to make each slot fit each lane. In this case, it is possible to make the multislot sheet with the desired number and widths of the slots. After use, the apparatus should be washed with detergent, rinsed with distilled water, and air dried at room temperature. We used the same apparatus over 20 times and still obtained good results. In summary, we reported the preparation and use of an apparatus for immunoblotting using a magnetic rubber sheet. This apparatus held a membrane tightly enough to prevent leaking of the antibody solution to neighboring lanes although it was prepared and used very easily. Acknowledgments. We thank Prof. Miyazaki (Nagoya University, Japan) for kindly giving us the anti-EF-2 and anti-EF-3 antibodies, and Prof. Ishikawa and Prof. Yoshinaga (Shizuoka University, Japan) for critical reading of the manuscript.
REFERENCES 1. Harlow, E., and Lane, D. (1988) in Antibodies: A Laboratory Manual, pp. 471–510, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. 2. Miyazaki, M., Uritani, M., Fujimura, K., Yamakatsu, H., Kageyama, T., and Takahashi, K. (1988) J. Biochem. 103, 508–521. 3. Laemmli, U. K. (1970) Nature 227, 680–685.
AP-Anal Bio