ANALYTICAL
BIOCHEMISTRY
(19%)
197,187-190
Standard Calibration Proteins for Western Blotting Obtained by Genetically Prepared Protein A Conjugates’ Christer
Lindbladh, Klaus Department of Pure and Applied
Received
March
Mosbach, Biochemistry,
and Leif Billow Chemical
Center,
of Lund,
P.O. Box 124, S-221 00 Lund,
Sweden
5, 1991
Genetically prepared protein A fusion proteins, having retained antibody binding capacity, were used to design different well-defined standard molecular weight marker proteins for Western blotting. The blotted marker proteins are developed at the same time and with the same reagents as the protein sample of interest. 0 1991 Academic Press, Inc.
Western blotting was first introduced by Towbin et al. immunological tool for the identification of various proteins. Detection of primary antibodies with specificity to a protein of interest can be achieved by different methods, including the use of enzyme-labeled or radiolabeled secondary antibodies (1). Alternatively, the secondary antibodies can be gold- or silver-labeled (2,3). However, it is often desirable to have calibration proteins with known molecular weights when the blotted sample is to be identified. Previously, this has been accomplished by removing one lane from the membrane containing different standard proteins and then staining this lane separately. This method often gives unreliable results due to potential shrinkage of the membrane in the staining procedure. Another problem that must be considered in staining the calibration proteins is the character of the membrane, anionic or cationic, as this is important when one is selecting a proper dye (4). To circumvent these problems prestained proteins (5) or biotinylated proteins (6) have been utilized as molecular weight standards for Western blotting. However, other investigators have pointed out serious disadvantages when using these techniques, such as deviation of the apparent molecular weight, up to 10% after conjugation with biotin (7), or (1) and has since become an important
1 This project and Agricultural velopment.
University
was supported by the Swedish Council for Forestry Research and the Swedish Board for Technical De-
0003-2697191 $3.00 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
poor resolution of the blotted prestained protein markers (6,8). In this study we have evaluated the possibilities of using different protein A fusion proteins, obtained by gene fusion, as calibration proteins for Western blotting. No extra procedures are needed to visualize the calibration proteins since the antibodies that are used to develop the sample will also bind unspecifically to the protein A moiety of the marker proteins, provided that either the primary or the secondary antibody used in the procedure has affinity to protein A. This latter requirement is often fulfilled simply because protein A binds immunoglobulins of many species (9). The use of genetically prepared fusion proteins also gives well-defined conjugates compared to the heterogeneous crosslinking products obtained by the conventional chemical coupling procedures between a protein and a marker molecule. Furthermore, calibration proteins produced by recombinant bacteria should be easy to produce at a low cost. MATERIALS
AND
METHODS
Plasmids and bacteria. Plasmid pCH 40, encoding alkaline phosphatase, was a gift from C. S. Hoffman (10). Plasmid pRITBT-E, encoding protein A, and plasmid pAB 1, encoding protein A/P-subunit of luciferase, have been described elsewhere (11). Restriction enzymes and T4-DNA ligase were purchased from Boehringer-Mannheim and all reactions were carried out as recommended by the manufacturer. Escherichia coli JM 105 (12) was used as host cell and LB-broth (13) as growth medium. Ampicillin (100 pg/ml) and isopropyl-@-D-thiogalactopyranoside (0.1 mM) were from Sigma and supplemented to the growth medium when necessary, Preparation of calibration proteins. Cells producing fusion protein were grown overnight at 37°C in LBbroth. The culture was centrifuged 5 min at 12,000g and 187
188
LINDBLADH,
MOSBACH,
resuspended in a sample buffer containing 0.25 M TrisHCl, pH 6.8, 6% SDS,’ 20% glycerol, 10% /3-mercaptoethanol, and bromophenol blue. The cell suspension was then incubated 3 min at 100°C before appropriate amounts of the different hybrid proteins were mixed. This mixture of cell extract, containing fusion proteins of different molecular weights, was used directly as standards for Western blotting. When necessary, the fusion protein was further purified by elution of the hybrid protein from a SDS-polyacrylamide gel. This was accomplished by homogenization of the gel slice containing the purified fusion protein and elution of the protein in 0.1% SDS, 50 mM Tris-HCl, pH 7.5, for 24 h at 25°C (14). Electrophoresis and Western blotting. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed according to Laemmli using a 10% polyacrylamide slab gel in a Tris-glycine, pH 8.3, discontinuous buffer system (15). Samples were transferred to a positively charged nylon membrane, Zeta-Probe (Bio-Rad), in a semidry electroblotter for 1 h at 0.8 mA/cm2, as recommended by the manufacturer (JKA-Biotech, Denmark). Nitrocellulose membranes functioned equally well but nylon membranes were selected because of their higher mechanical strength. The electroblot was blocked for 5 min employing 2% Tween 20 dissolved in TBS (0.05 M Tris-HCl, pH 7.4,0.15 M NaCl), at ambient temperature. After washing for 1 min in TBS, the membrane was incubated overnight in horseradish peroxidase (HRP)-conjugated anti-guinea pig rabbit serum (Dakopatts), previously dissolved 1:300 in TBS, 0.5% Tween 20. Finally, the blot was washed three times for 5 min using fresh TBS before subsequent visualization of the protein bands by the substrates H,O, and 4-chloro1-naphthol, as recommended by Bio-Rad’s instructions. Anti-porcine insulin guinea pig serum (1:300) obtained from Sigma and anti-calf alkaline phosphatase rabbit serum (1:300) from Dakopatts were used as primary antibodies (overnight incubation) in order to evaluate whether the presence of antibodies, with or without antigenic determinants for the HRP-conjugated secondary antibody, affected the development of the calibration proteins described above. Thus, incubation with anti-calf alkaline phosphatase rabbit serum, as primary antibodies, was used to determine whether it was possible to mask protein A from binding to HRP-conjugated secondary antibodies. RESULTS
AND
DISCUSSION
Construction and production of calibration proteins. The construction of a number of protein A gene ’ Abbreviations used: HRP, acrylamide gel electrophoresis; Tris-buffered saline.
horseradish peroxidase; SDS, sodium dodecyl
PAGE, sulfate;
polyTBS,
AND
BijLOW
fusion vectors has been described (16,17) and some of them, pRIT2 and pRIT5, are also commercially available. One purpose of using these vectors has been to facilitate the purification or the immobilization of a protein of interest. Recently, we have also described the use of a protein Ailuciferase conjugate for bioluminescent immunoassays (11). When utilizing these different protein A conjugates we realized that some of them could also be most valuable for Western blotting purposes, as well-defined molecular weight marker proteins. The fusion proteins used to exemplify this technique have molecular weights between 30 and 73 kDa. The individual proteins primarily selected were protein A, protein A/luciferase &subunit, and protein A/alkaline phosphatase. The protein A/alkaline phosphatase fusion protein was obtained by the insertion of a 2900-bp Pst I fragment from plasmid pCH 40, encoding alkaline phosphatase, into the 3’-terminal end of the structural gene encoding protein A in plasmid pRIT5. The different fusion proteins obtained from E. coli, harboring the described plasmids, were recovered from an overnight culture. The choice of suitable fusion proteins is partly dependent on their stability. Among the standard proteins produced in this way the protein A/ alkaline phosphatase conjugate had to be purified before blotting, as outlined under Materials and Methods, because of minor proteolytic degradation in the host cell. Purification from SDS-PAGE is illustrated in Fig. 1, which shows that the proteolytic degradation products can effectively be removed by a simple one-step procedure. Appropriate amounts of crude homogenates containing the desired fusion protein and the purified fusion protein were mixed and dissolved in sample buffer. The protein mixtures were stable for several months when stored at -20°C. The use of protein A fusion proteins as molecular weight markers in Western blotting is not limited to the range of 30 to 73 kDa. Protein A is composed of five homologous domains, each having IgG binding capacity. For instance, employment of only two domains of protein A (18) can be utilized to prepare markers with molecular weights less than 30 kDa. The upper limit can easily be extended by using a larger protein than alkaline phosphatase, e.g. fl-galactosidase (E. coli) having M, = 116,000 (19). Recently, other protein A fusion proteins have also been described (20,21). In our attempts to prepare calibration proteins with intermediate molecular weights we also deleted a l.l-kb DNA fragment from the 5’-terminal end of the alkaline phosphatase gene before fusion to protein A. Obviously, this fusion protein did not form stable structure and was therefore rapidly degraded. This observation and other investigations on proteolytic degradation of abnormal proteins in uiuo (22) suggest that the best results will be obtained when either entire proteins or stably folded protein do-
WESTERN
BLOTTING-GENETICALLY
mains are used in the design of molecular weight markers. Western blotting. Positively charged Zeta-Probe nylon membrane was used in the Western blotting experiments. It has many excellent properties as a blotting matrix but has the disadvantage of being difficult to nonspecifically stain when one is visualizing the blotted proteins (4). This problem is avoided when the protein A conjugates are used. Figure 2 shows the molecular weights of the different fusion proteins in a typical blotting experiment. The sample mixture does not produce any background that interferes with the interpretation of the individual positions of the calibration proteins. The blotted proteins were also incubated with anti-calf alkaline phosphatase rabbit serum in order to evaluate whether the unspecific binding of this antibody to the protein A moiety of the calibration proteins interfered with the second incubation of HRP-conjugated antibodies. Despite the utilization of a high concentration of the primary antibody (1:300) in an overnight incubation and the lack of affinity between the primary antibody and the secondary HRP-conjugated antibody, it was not possible to mask the protein A chimeric calibration proteins from binding the secondary HRP-conjugated antibody. Thus, no extra step needs to be included in order
PREPARED
CALIBRATION
I
73 kDa
kDa
II
Ill
IV
-
63 kDa -
30
189
PROTEINS
-
l
FIG. 2. Western blotting of the engineered calibration proteins. Lane I, three fusion proteins mixed together. From the bottom: whole cell extract of E. coli producing protein A, 30 kDa; whole cell extract of E. coli producing protein Ajluciferase &subunit, 63 kDa; purified protein A/alkaline phosphatase, 73 kDa. Lane II, whole-cell extract of E. coli producing protein A, 30 kDa. Lane III, whole-cell extract of E. coli producing protein A/luciferase @-subunit, 63 kDa. Lane IV, purified protein A/alkaline phosphatase, 73 kDa. Full details are given under Materials and Methods.
to develop the sample of interest and the standard calibration proteins provided that protein A has affinity to either the primary or the secondary antibody, or both. Recently, protein G fusion vectors have been constructed, thus implying that the described technique could be extended to a broader range of immunoglobulin species, including monoclonal antibodies (23). In this paper we have described a novel method to prepare calibration proteins suitable for Western blotting. Conjugation of protein A with other proteins by use of gene fusion is often preferable to the employment of conventional chemical crosslinking methods since well-defined and homogeneous marker proteins can easily be produced. Moreover, gene fusion is not limited to blotting procedures but should be an attractive alternative for the production of standard marker proteins suitable for any form of electrophoresis or chromatography. REFERENCES FIG. 1. Western blotting of different elution fractions of the fusion protein, protein A/alkaline phosphatase. Protein fractions were eluted from SDS-polyacrylamide gel slices containing the desired fusion protein. Lane I, a major degradation product from the fusion protein, purified by the method outlined under Materials and Methods. Lane II, purified protein A/alkaline phosphatase. Lane III, whole-cell extract of E. coli producing protein A/alkaline phosphatase. The fusion protein is subjected to proteolytic degradation in uiuo.
1. Towbin, H., Staehelin, Sci. USA 76,4350-4354.
T., and Gordon,
J. (1979)
Proc. Natl.
Acczd.
2. Moeremans, M., Daneels, G., Van Dijck, A., Langanger, G., and De Mey, J. (1984) J. Zmmunol. Methods 74, 353-360. 3. Daneels, G., Moeremans, M., Raeymaeker, M. D., and De Mey, J. (1986) J. Zmmunol. Methods 89,89-91. 4. Hughes, J. H., Mack, K., and Hamparian, V. V. (1966) And. Bio&em. 173,18-25.
190
LINDBLADH,
5. Parkinson, 121-126. 6. Della-Penna,
D., and
Anal. Biochem. 141,
J. D. (1984)
D., Christoffersen,
Anal. B&hem. 7. Neumaier,
Redshaw,
MOSBACH,
R. E., and Bennett,
A. B. (1986)
162.329-332.
M., Fenger,
U., and Wagener,
Anal. Biochem.
C. (1986)
156,76-80. 8. Tsang,
V. C. W.,
Hancock,
K., and
Simons,
A. R. (1984)
Anal.
Biochem. 143,304-307. 9. Langone, 10. Hoffman, 82,5307-5111.
C., Mosbach, K., and Billow, L. (1991) J. Immunol. Methods 137,199-207. 12. Messing, J. (1983) in Methods in Enzymology (Wu, R., Grossman, 11. Lindbladh,
86.
BULOW
15. Laemmli,
101, pp. 20-78,
Academic
Press,
U. K. (1970)
Genetics, NY.
Cold Spring
Anal. Biochem. 109,76-
L. and
Uhlen,
M.
17. Uhlen, M., Nilsson, B., Guss, B., Lindberg, and Philipson, L. (1983) Gene 23,369-378. 18. Nilsson, Holmgren,
(1985) M.,
EMBO J. 4, Gatenbeck,
S.,
B., Moks, B., Jansson, B., Abrahmsen, L., Elmblad, A., E., Henrichson, C., Jones, A. T., and Uhlen, M. (1987) Eng. 1, 107-113.
19. Casadaban, M. J., Martinez-Arias, A., Shapira, S. K., and Chou, J. (1983) in Methods in Enzymology (Wu, R., Grossman, L., and Moldave, K., Eds.), Vol. 100, pp. 293-308, Academic Press, San Diego, CA. 20. Kim,
J.-H.,
21. Wada, (1990)
M.,
and Weaver, Kasumimoto,
F. (1988) T., Miki,
Gene
68,315-321.
T., Osada,
H., and Kato,
S.
J. Biotechnol. 13, 325-334.
22. Goldberg, Molecular Harbor, (1980)
Nature 227,680-685.
16. Nilsson, B., Abrahmsen, 1075-1080.
Protein
Adv. Immunol. 32,157-241. C. S., and Wright, A. (1985) Proc. Natl. Acad. Sci. USA
J. J. (1982)
L., and Moldave, K., Eds.), Vol. San Diego, CA. 13. Miller, J. (1972) Experiments in Harbor Laboratory, Cold Spring 14. Hager, D. A., and Burgess, R. R.
AND
A. L., and St. John,
A.
C. (1976)
Ann. Rev. B&hem.
45,747-803. 23. Guss,
B., Eliasson, Jornvall, H., Flock, 1567-1575.
M., J.-I.,
Olsson, A., and Lindberg,
Uhlen, M., M. (1986)
Frej,
A.-K.,
EMBO J. 5,
BIOCHEMISTRY
(19%)
197,187-190
Standard Calibration Proteins for Western Blotting Obtained by Genetically Prepared Protein A Conjugates’ Christer
Lindbladh, Klaus Department of Pure and Applied
Received
March
Mosbach, Biochemistry,
and Leif Billow Chemical
Center,
of Lund,
P.O. Box 124, S-221 00 Lund,
Sweden
5, 1991
Genetically prepared protein A fusion proteins, having retained antibody binding capacity, were used to design different well-defined standard molecular weight marker proteins for Western blotting. The blotted marker proteins are developed at the same time and with the same reagents as the protein sample of interest. 0 1991 Academic Press, Inc.
Western blotting was first introduced by Towbin et al. immunological tool for the identification of various proteins. Detection of primary antibodies with specificity to a protein of interest can be achieved by different methods, including the use of enzyme-labeled or radiolabeled secondary antibodies (1). Alternatively, the secondary antibodies can be gold- or silver-labeled (2,3). However, it is often desirable to have calibration proteins with known molecular weights when the blotted sample is to be identified. Previously, this has been accomplished by removing one lane from the membrane containing different standard proteins and then staining this lane separately. This method often gives unreliable results due to potential shrinkage of the membrane in the staining procedure. Another problem that must be considered in staining the calibration proteins is the character of the membrane, anionic or cationic, as this is important when one is selecting a proper dye (4). To circumvent these problems prestained proteins (5) or biotinylated proteins (6) have been utilized as molecular weight standards for Western blotting. However, other investigators have pointed out serious disadvantages when using these techniques, such as deviation of the apparent molecular weight, up to 10% after conjugation with biotin (7), or (1) and has since become an important
1 This project and Agricultural velopment.
University
was supported by the Swedish Council for Forestry Research and the Swedish Board for Technical De-
0003-2697191 $3.00 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
poor resolution of the blotted prestained protein markers (6,8). In this study we have evaluated the possibilities of using different protein A fusion proteins, obtained by gene fusion, as calibration proteins for Western blotting. No extra procedures are needed to visualize the calibration proteins since the antibodies that are used to develop the sample will also bind unspecifically to the protein A moiety of the marker proteins, provided that either the primary or the secondary antibody used in the procedure has affinity to protein A. This latter requirement is often fulfilled simply because protein A binds immunoglobulins of many species (9). The use of genetically prepared fusion proteins also gives well-defined conjugates compared to the heterogeneous crosslinking products obtained by the conventional chemical coupling procedures between a protein and a marker molecule. Furthermore, calibration proteins produced by recombinant bacteria should be easy to produce at a low cost. MATERIALS
AND
METHODS
Plasmids and bacteria. Plasmid pCH 40, encoding alkaline phosphatase, was a gift from C. S. Hoffman (10). Plasmid pRITBT-E, encoding protein A, and plasmid pAB 1, encoding protein A/P-subunit of luciferase, have been described elsewhere (11). Restriction enzymes and T4-DNA ligase were purchased from Boehringer-Mannheim and all reactions were carried out as recommended by the manufacturer. Escherichia coli JM 105 (12) was used as host cell and LB-broth (13) as growth medium. Ampicillin (100 pg/ml) and isopropyl-@-D-thiogalactopyranoside (0.1 mM) were from Sigma and supplemented to the growth medium when necessary, Preparation of calibration proteins. Cells producing fusion protein were grown overnight at 37°C in LBbroth. The culture was centrifuged 5 min at 12,000g and 187
188
LINDBLADH,
MOSBACH,
resuspended in a sample buffer containing 0.25 M TrisHCl, pH 6.8, 6% SDS,’ 20% glycerol, 10% /3-mercaptoethanol, and bromophenol blue. The cell suspension was then incubated 3 min at 100°C before appropriate amounts of the different hybrid proteins were mixed. This mixture of cell extract, containing fusion proteins of different molecular weights, was used directly as standards for Western blotting. When necessary, the fusion protein was further purified by elution of the hybrid protein from a SDS-polyacrylamide gel. This was accomplished by homogenization of the gel slice containing the purified fusion protein and elution of the protein in 0.1% SDS, 50 mM Tris-HCl, pH 7.5, for 24 h at 25°C (14). Electrophoresis and Western blotting. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed according to Laemmli using a 10% polyacrylamide slab gel in a Tris-glycine, pH 8.3, discontinuous buffer system (15). Samples were transferred to a positively charged nylon membrane, Zeta-Probe (Bio-Rad), in a semidry electroblotter for 1 h at 0.8 mA/cm2, as recommended by the manufacturer (JKA-Biotech, Denmark). Nitrocellulose membranes functioned equally well but nylon membranes were selected because of their higher mechanical strength. The electroblot was blocked for 5 min employing 2% Tween 20 dissolved in TBS (0.05 M Tris-HCl, pH 7.4,0.15 M NaCl), at ambient temperature. After washing for 1 min in TBS, the membrane was incubated overnight in horseradish peroxidase (HRP)-conjugated anti-guinea pig rabbit serum (Dakopatts), previously dissolved 1:300 in TBS, 0.5% Tween 20. Finally, the blot was washed three times for 5 min using fresh TBS before subsequent visualization of the protein bands by the substrates H,O, and 4-chloro1-naphthol, as recommended by Bio-Rad’s instructions. Anti-porcine insulin guinea pig serum (1:300) obtained from Sigma and anti-calf alkaline phosphatase rabbit serum (1:300) from Dakopatts were used as primary antibodies (overnight incubation) in order to evaluate whether the presence of antibodies, with or without antigenic determinants for the HRP-conjugated secondary antibody, affected the development of the calibration proteins described above. Thus, incubation with anti-calf alkaline phosphatase rabbit serum, as primary antibodies, was used to determine whether it was possible to mask protein A from binding to HRP-conjugated secondary antibodies. RESULTS
AND
DISCUSSION
Construction and production of calibration proteins. The construction of a number of protein A gene ’ Abbreviations used: HRP, acrylamide gel electrophoresis; Tris-buffered saline.
horseradish peroxidase; SDS, sodium dodecyl
PAGE, sulfate;
polyTBS,
AND
BijLOW
fusion vectors has been described (16,17) and some of them, pRIT2 and pRIT5, are also commercially available. One purpose of using these vectors has been to facilitate the purification or the immobilization of a protein of interest. Recently, we have also described the use of a protein Ailuciferase conjugate for bioluminescent immunoassays (11). When utilizing these different protein A conjugates we realized that some of them could also be most valuable for Western blotting purposes, as well-defined molecular weight marker proteins. The fusion proteins used to exemplify this technique have molecular weights between 30 and 73 kDa. The individual proteins primarily selected were protein A, protein A/luciferase &subunit, and protein A/alkaline phosphatase. The protein A/alkaline phosphatase fusion protein was obtained by the insertion of a 2900-bp Pst I fragment from plasmid pCH 40, encoding alkaline phosphatase, into the 3’-terminal end of the structural gene encoding protein A in plasmid pRIT5. The different fusion proteins obtained from E. coli, harboring the described plasmids, were recovered from an overnight culture. The choice of suitable fusion proteins is partly dependent on their stability. Among the standard proteins produced in this way the protein A/ alkaline phosphatase conjugate had to be purified before blotting, as outlined under Materials and Methods, because of minor proteolytic degradation in the host cell. Purification from SDS-PAGE is illustrated in Fig. 1, which shows that the proteolytic degradation products can effectively be removed by a simple one-step procedure. Appropriate amounts of crude homogenates containing the desired fusion protein and the purified fusion protein were mixed and dissolved in sample buffer. The protein mixtures were stable for several months when stored at -20°C. The use of protein A fusion proteins as molecular weight markers in Western blotting is not limited to the range of 30 to 73 kDa. Protein A is composed of five homologous domains, each having IgG binding capacity. For instance, employment of only two domains of protein A (18) can be utilized to prepare markers with molecular weights less than 30 kDa. The upper limit can easily be extended by using a larger protein than alkaline phosphatase, e.g. fl-galactosidase (E. coli) having M, = 116,000 (19). Recently, other protein A fusion proteins have also been described (20,21). In our attempts to prepare calibration proteins with intermediate molecular weights we also deleted a l.l-kb DNA fragment from the 5’-terminal end of the alkaline phosphatase gene before fusion to protein A. Obviously, this fusion protein did not form stable structure and was therefore rapidly degraded. This observation and other investigations on proteolytic degradation of abnormal proteins in uiuo (22) suggest that the best results will be obtained when either entire proteins or stably folded protein do-
WESTERN
BLOTTING-GENETICALLY
mains are used in the design of molecular weight markers. Western blotting. Positively charged Zeta-Probe nylon membrane was used in the Western blotting experiments. It has many excellent properties as a blotting matrix but has the disadvantage of being difficult to nonspecifically stain when one is visualizing the blotted proteins (4). This problem is avoided when the protein A conjugates are used. Figure 2 shows the molecular weights of the different fusion proteins in a typical blotting experiment. The sample mixture does not produce any background that interferes with the interpretation of the individual positions of the calibration proteins. The blotted proteins were also incubated with anti-calf alkaline phosphatase rabbit serum in order to evaluate whether the unspecific binding of this antibody to the protein A moiety of the calibration proteins interfered with the second incubation of HRP-conjugated antibodies. Despite the utilization of a high concentration of the primary antibody (1:300) in an overnight incubation and the lack of affinity between the primary antibody and the secondary HRP-conjugated antibody, it was not possible to mask the protein A chimeric calibration proteins from binding the secondary HRP-conjugated antibody. Thus, no extra step needs to be included in order
PREPARED
CALIBRATION
I
73 kDa
kDa
II
Ill
IV
-
63 kDa -
30
189
PROTEINS
-
l
FIG. 2. Western blotting of the engineered calibration proteins. Lane I, three fusion proteins mixed together. From the bottom: whole cell extract of E. coli producing protein A, 30 kDa; whole cell extract of E. coli producing protein Ajluciferase &subunit, 63 kDa; purified protein A/alkaline phosphatase, 73 kDa. Lane II, whole-cell extract of E. coli producing protein A, 30 kDa. Lane III, whole-cell extract of E. coli producing protein A/luciferase @-subunit, 63 kDa. Lane IV, purified protein A/alkaline phosphatase, 73 kDa. Full details are given under Materials and Methods.
to develop the sample of interest and the standard calibration proteins provided that protein A has affinity to either the primary or the secondary antibody, or both. Recently, protein G fusion vectors have been constructed, thus implying that the described technique could be extended to a broader range of immunoglobulin species, including monoclonal antibodies (23). In this paper we have described a novel method to prepare calibration proteins suitable for Western blotting. Conjugation of protein A with other proteins by use of gene fusion is often preferable to the employment of conventional chemical crosslinking methods since well-defined and homogeneous marker proteins can easily be produced. Moreover, gene fusion is not limited to blotting procedures but should be an attractive alternative for the production of standard marker proteins suitable for any form of electrophoresis or chromatography. REFERENCES FIG. 1. Western blotting of different elution fractions of the fusion protein, protein A/alkaline phosphatase. Protein fractions were eluted from SDS-polyacrylamide gel slices containing the desired fusion protein. Lane I, a major degradation product from the fusion protein, purified by the method outlined under Materials and Methods. Lane II, purified protein A/alkaline phosphatase. Lane III, whole-cell extract of E. coli producing protein A/alkaline phosphatase. The fusion protein is subjected to proteolytic degradation in uiuo.
1. Towbin, H., Staehelin, Sci. USA 76,4350-4354.
T., and Gordon,
J. (1979)
Proc. Natl.
Acczd.
2. Moeremans, M., Daneels, G., Van Dijck, A., Langanger, G., and De Mey, J. (1984) J. Zmmunol. Methods 74, 353-360. 3. Daneels, G., Moeremans, M., Raeymaeker, M. D., and De Mey, J. (1986) J. Zmmunol. Methods 89,89-91. 4. Hughes, J. H., Mack, K., and Hamparian, V. V. (1966) And. Bio&em. 173,18-25.
190
LINDBLADH,
5. Parkinson, 121-126. 6. Della-Penna,
D., and
Anal. Biochem. 141,
J. D. (1984)
D., Christoffersen,
Anal. B&hem. 7. Neumaier,
Redshaw,
MOSBACH,
R. E., and Bennett,
A. B. (1986)
162.329-332.
M., Fenger,
U., and Wagener,
Anal. Biochem.
C. (1986)
156,76-80. 8. Tsang,
V. C. W.,
Hancock,
K., and
Simons,
A. R. (1984)
Anal.
Biochem. 143,304-307. 9. Langone, 10. Hoffman, 82,5307-5111.
C., Mosbach, K., and Billow, L. (1991) J. Immunol. Methods 137,199-207. 12. Messing, J. (1983) in Methods in Enzymology (Wu, R., Grossman, 11. Lindbladh,
86.
BULOW
15. Laemmli,
101, pp. 20-78,
Academic
Press,
U. K. (1970)
Genetics, NY.
Cold Spring
Anal. Biochem. 109,76-
L. and
Uhlen,
M.
17. Uhlen, M., Nilsson, B., Guss, B., Lindberg, and Philipson, L. (1983) Gene 23,369-378. 18. Nilsson, Holmgren,
(1985) M.,
EMBO J. 4, Gatenbeck,
S.,
B., Moks, B., Jansson, B., Abrahmsen, L., Elmblad, A., E., Henrichson, C., Jones, A. T., and Uhlen, M. (1987) Eng. 1, 107-113.
19. Casadaban, M. J., Martinez-Arias, A., Shapira, S. K., and Chou, J. (1983) in Methods in Enzymology (Wu, R., Grossman, L., and Moldave, K., Eds.), Vol. 100, pp. 293-308, Academic Press, San Diego, CA. 20. Kim,
J.-H.,
21. Wada, (1990)
M.,
and Weaver, Kasumimoto,
F. (1988) T., Miki,
Gene
68,315-321.
T., Osada,
H., and Kato,
S.
J. Biotechnol. 13, 325-334.
22. Goldberg, Molecular Harbor, (1980)
Nature 227,680-685.
16. Nilsson, B., Abrahmsen, 1075-1080.
Protein
Adv. Immunol. 32,157-241. C. S., and Wright, A. (1985) Proc. Natl. Acad. Sci. USA
J. J. (1982)
L., and Moldave, K., Eds.), Vol. San Diego, CA. 13. Miller, J. (1972) Experiments in Harbor Laboratory, Cold Spring 14. Hager, D. A., and Burgess, R. R.
AND
A. L., and St. John,
A.
C. (1976)
Ann. Rev. B&hem.
45,747-803. 23. Guss,
B., Eliasson, Jornvall, H., Flock, 1567-1575.
M., J.-I.,
Olsson, A., and Lindberg,
Uhlen, M., M. (1986)
Frej,
A.-K.,
EMBO J. 5,
