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Heat-mediated, ultra-rapid electrophoretic transfer of high and low molecular weight proteins to nitrocellulose membranes
Journal of Immunological Methods 266 (2002) 127 – 133 www.elsevier.com/locate/jim
Heat-mediated, ultra-rapid electrophoretic transfer of high and low molecular weight proteins to nitrocellulose membranes Biji T. Kurien a,*, R. Hal Scofield a,b,c a
Arthritis and Immunology Program, Oklahoma Medical Research Foundation, 825 NE 13th Street, Oklahoma City, OK 73104, USA b Department of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA c Department of Veterans Affairs Medical Center, Oklahoma City, OK 73104, USA Received 10 December 2001; received in revised form 8 April 2002; accepted 9 April 2002
Abstract Here, we report an ultra-rapid method for the transfer of high and low molecular weight proteins to nitrocellulose membranes following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In this procedure, the electrotransfer was performed with heated (70 – 75 jC) normal transfer buffer from which methanol had been omitted. Complete transfer of high and low molecular weight proteins (a purified protein, molecular weight protein standards and proteins from a human tissue extract) could be carried out in 10 min for a 0.75-mm, 7% SDS-PAGE gel. For 10% and 12.5% gels (0.75 mm), the corresponding time was 15 min. In the case of 1.5-mm gels, a complete transfer could be carried out in 20 min for 7%, 10% and 12.5% gels. The permeability of the gel is increased by heat, such that the proteins trapped in the polyacrylamide gel matrix can be easily transferred to the membrane. When the heat-mediated transfer method was compared with a conventional transfer protocol, under similar conditions, we found that the latter method transferred minimal low molecular weight proteins while retaining most of the high molecular weight proteins in the gel. In summary, this procedure is very rapid, avoids the use of methanol and is particularly useful for the transfer of high molecular weight proteins. D 2002 Elsevier Science B.V. All rights reserved. Keywords: SDS-PAGE; Western blotting; Nitrocellulose
1. Introduction Transfer of proteins from sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels to nitrocellulose or polyvinylidene difluoride membranes have been achieved in three different ways: (a) simple diffusion (Kurien and Scofield, 1997); vac*
Corresponding author. Tel.: +1-405-271-7394; fax: +1-405271-4110. E-mail address: [email protected] (B.T. Kurien).
uum-assisted solvent flow (Peferoen et al., 1982); and (c) electrophoretic elution (Towbin et al., 1979). There is considerable interest in diffusion-mediated transfer of proteins, since Kurien and Scofield (1997) showed that multiple immunoblots can be generated after nonelectrophoretic bidirectional transfer of a single SDSPAGE gel with multiple antigens. The lifts from SDSPAGE gels, for immunoblotting, using this method are particularly useful in identification of proteins by mass spectrometry (Kurien et al., 2000; Kurien et al., 2001a,b). However, electrophoretic elution, widely
0022-1759/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 2 - 1 7 5 9 ( 0 2 ) 0 0 1 0 3 - 5
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B.T. Kurien, R.H. Scofield / Journal of Immunological Methods 266 (2002) 127–133
used in most laboratories, is still the method of choice for transfer of proteins to membranes. Electrophoretic transfer of proteins to nitrocellulose, subsequent to separation by SDS-PAGE, is a fundamental step prior to detection of specific proteins with specific antibodies (Towbin et al., 1979). The Western transfer of proteins normally takes about 2 –4 h at about 70 V or an overnight transfer at 30 V. High molecular weight proteins are often stubbornly resistant to transfer (Bolt and Mahoney, 1997; Kurien and Scofield, unpublished observation) in spite of these prolonged runs. This problem is accentuated when higher percentage gels are used. Prolonged electrotransfer (16 – 20 h) at high current density coupled with inclusion of 0.01% sodium dodecyl sulfate, to enhance protein elution, has been used to efficiently transfer high-molecular weight proteins (Otter et al., 1987). Here, we show that both low and high molecular weight proteins can be very efficiently transferred to nitrocellulose membrane in a very short period of time using heated, normal transfer buffer without methanol.
The cells were lysed by sonication using a Branson sonicator using setting 4 in SDS-PAGE lysis buffer and spun at 10,000 g for 10 min. An aliquot of the supernatant was analyzed by SDS-PAGE, followed by immunoblotting. 2.2.2. SDS-PAGE SDS-PAGE was carried out according to Laemmli (1970). Bovine 60 kDa Ro (1 Ag/strip) and prestained protein standards (5 Al/lane) were separated on 7%, 10% or 12.5% gels (0.75- or 1.5mm thickness) and transferred to nitrocellulose. Protein standards were used at 10 Al/lane for the experiments that compared the efficiency of the heat transfer method with that obtained using a conventional method. 2.2.3. Electrophoretic transfer Conventional electro-transfer of proteins separated on gradient and regular gels was carried out at 4 jC using standard transfer buffer (25 mM Tris, 190 mM Glycine, 20% methanol) and compared with heatmediated electrophoretic transfer under similar conditions.
2. Materials and methods 2.1. Materials Nitrocellulose membranes were purchased from Gelman Sciences. Purified bovine 60 kDa Ro (Deutscher et al., 1988; Yamagata et al., 1984) was a gift from Immunovision (Springdale, AK). BenchMark pre-stained molecular weight standards were obtained from Gibco BRL (Bethesda, MD). All other chemicals used were of reagent grade. Western blot transfer apparatus, possessing the capability of circulating hot or cold water at the base, was purchased from Hoeffer Scientific Instruments (Pharmacia/Amersham). A refrigerator bath/circulating water bath (Endocal RT110), capable of circulating hot or cold water was from Neslab, Portsmouth, NH. Branson Sonifier Cell Disruptor 185 was from VWR Scientific, (Boston, MA). 2.2. Methods 2.2.1. Preparation of HeLa cell lysate Freshly cultured HeLa cells were harvested and washed twice with phosphate-buffered saline, pH 7.4.
2.2.4. Heat-mediated electrophoretic transfer The transfer was carried out in the absence of methanol, using 25 mM Tris and 190 mM glycine buffer. The buffer was heated to 70– 75 jC in a 1-l beaker that was covered with clear plastic wrap. The hot buffer was transferred to a Hoeffer transfer apparatus, fabricated with the capability of circulating hot water (70 –75 jC) at the base of the apparatus (the hot water being supplied by a re-circulating water bath which should be turned on at least 30 min before completion of the SDS PAGE procedure so as to allow the water to attain the required temperature). This step was carried out after the gel transfer sandwich had been assembled. To assemble the gel sandwich, the nitrocellulose was moistened with the same buffer, taking about 10 min to be moistened completely. (Alternately, the nitrocellulose membrane can be moistened almost instantaneously with regular transfer buffer that contains methanol.) Preferably, the buffer used to assemble the sandwich should be at room temperature or just slightly warm. (Using the hot buffer to assemble the sandwich causes the 3 M filter paper, used as a part of the gel sandwich, to disinte-
B.T. Kurien, R.H. Scofield / Journal of Immunological Methods 266 (2002) 127–133
grate partially). The cassette with the gel-membrane sandwich was placed in the transfer apparatus. The transfer apparatus was placed on a Corning PC-351 magnetic stirrer. A magnetic stir bar was used to circulate the buffer inside the transfer apparatus. The transfer was carried out at 40 V for periods ranging from 10 to 20 min, depending upon the gel used, using a FB300 power supply from Fisher Scientific (Houston, TX, USA). 2.2.5. Immunoblotting Standard immunoblotting was carried out (Towbin et al., 1979). Human systemic lupus erythematosus (SLE) sera with autoantibodies against 60 kDa Ro, 48 kDa La, 52 kDa Ro, Sm and nuclear ribonucleoprotein autoantigens (Harley and Scofield, 1991; Scofield et al., 1999) were used to identify the respective antigens transferred to nitrocellulose from various gels.
3. Results We have used purified protein, prestained molecular weight standards and a human cell extract to
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determine the efficiency of heat-mediated electroblotting. In order to determine the efficiency of heatmediated transfer of proteins, gels of two different thicknesses—0.75 and 1.5 mm—and gels with three different amounts of acrylamide (7%, 10% and 12.5%) were used in this study. In addition, transfer of proteins from a 4– 20% gradient SDS-PAGE gel and a 12.5% (1.5 mm) regular gel was also investigated and compared to that obtained using a conventional transfer method under similar conditions. Bovine 60 kDa Ro (lane 2 in Figs. 1 and 2) and prestained protein standards (Lane 4 in Figs. 1 and 2) could be transferred entirely with respect to gels with different thicknesses and polyacrylamide amounts. It took about 10 min to transfer the entire pre-stained protein marker (5 Al of the marker) from a 0.75 mm, 7% gel (Fig. 1 A, lane 4). All the protein markers, ranging from 184 to 9 kDa, could be transferred to membranes from 7%, 10% and 12.5% gels (Fig. 1B,C, lane 4) in 15 min. The post-transfer polyacrylamide gels were clean, without any sign of residual non-transferred protein markers. It took 20 min to transfer all the protein markers in the case of the 1.5-mm gels (7%, 10% and 12.5% gels) (Fig. 2, lane 4).
Fig. 1. Western blot transfer and immunoblotting of bovine 60 kDA Ro and prestained molecular weight standards using 7%, 10% and 12.5% gels (0.75 mm gels). (A, B, C) Proteins on a 7%, 10% and 12.5% SDS-PAGE gels respectively transferred as mentioned in Materials and methods. Lane 1—anti-60 kDa negative control; lane 2—anti-Ro positive control; lane 3—conjugate control; lane 4—prestained molecular weight standards.
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Fig. 2. Western blot transfer and immunoblotting of bovine 60 kDa Ro and prestained molecular weight standards using 7%, 10% and 12.5% gels (1.5 mm gels). (A, B, C) Proteins on a 7%, 10% and 12.5% SDS-PAGE gels transferred as mentioned in Materials and methods. Lane 1—anti-60 kDa negative control; lane 2—anti-Ro positive control; lane 3—conjugate control; lane 4—prestained molecular weight standards.
To compare the efficiency of the transfer process using this method to that obtained with a conventional method, we carried out two experiments. In the first experiment, we analyzed pre-stained protein ladder and purified 60 kDa Ro on a 12.5% SDS-PAGE gel. One half of the gel, containing the marker (10 Al) and 60 kDa Ro (4 Ag/well), was transferred using our method. The other half, containing equal amount of the marker (10 Al) and 60 kDa Ro (4 Ag/well) was transferred, using a conventional transfer procedure as mentioned in Materials and methods. Similarly, equal volumes of marker and HeLa cell extract were analyzed on a 4 – 20% pre-cast gradient gel. One half of the gel, containing the HeLa cell extract and the protein marker, was transferred using our method and the other half, containing the same amount of the extract and marker, with a conventional method. Fig. 3 shows the Fast Green stained nitrocellulose membranes obtained after transfer of protein using our method (right) and a conventional method (left) from a 4 – 20% gradient gel. The transfer of the prestained molecular weight standards and the proteins from the lysate was very efficient with the heat transfer method compared to the conventional method. The low molecular weight markers and low
molecular weight proteins in the lysate have been barely transferred after 20 min of conventional transfer, while higher molecular weight proteins have not been transferred. Fig. 4A shows purified 60 kDa Ro and the markers transferred with the new heat method (left) compared to that obtained with the conventional method (right) using a 12.5% gel. An anti-60 kDa Ro containing lupus sera binds strongly to 60 kDa Ro in the blot on the left, much stronger than it does on the right. In addition, molecular weight markers (lane 5, left) have been transferred efficiently, while only the lower molecular weight proteins were just barely transferred (lane 5, right). This clearly shows that the transfer was more efficient using the heat transfer compared to the regular transfer. Fig. 4B shows the immunoblot of HeLa extract obtained after transfer, from a 4– 20% gradient gel, using our method (left) and that obtained using a conventional method (right). Both the gels were transferred under similar conditions. Anti-60 kDa Ro, antiLa, anti-52 kDa Ro and anti-Sm/nRNP containing lupus sera (lanes 3 – 6, respectively; left) bind very strongly to their respective antigens on the blot obtained using the heat transfer method. Whereas
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could just barely be transferred using the conventional method (lane 7, right). All the markers could be efficiently transferred using the heat transfer procedure (lane 7, left). This, once again, shows that the transfer of proteins is partial after a 20-min transfer using a conventional means of transferring protein from a gel to the membrane.
Fig. 3. Fast Green staining of proteins from a HeLa cell extract transferred to nitrocellulose membrane by a conventional method (left) and using the heat transfer method (right) from a 4 – 20% SDSPAGE gradient gel. Lane 1—HeLa cell extract; lane 2—10 Al of prestained molecular weight marker.
only the anti-Ro sera and anti-Sm/nRNP sera bind, in the case of the blot that had been transferred using a conventional method, and the binding is weak (lanes 3 and 6, right). In addition, only the low molecular weight protein markers (16.8, 21.7 and 28.4 kDa)
Fig. 4. Immunoblots of purified 60 kDa Ro and proteins derived from a HeLa cell extract transferred to nitrocellulose membrane using the heat transfer method (left) and by a conventional method (right). (A) Purified 60 kDa Ro autoantigen immunoblot obtained from a 12.5% SDS-PAGE gel using the heat transfer method (left) and by a conventional method (right). Lane 1—conjugate control; lanes 2 and 3—normal controls; lane 4—anti-60 kDa Ro SLE sera; lane 5—prestained protein molecular weight standards. (B) HeLa cell extract immunoblot obtained from a 4 – 20% gradient SDSPAGE gel using the heat transfer method (left) and a conventional transfer method (right). Lane1—conjugate control; lane 2—normal control; lane 3—anti-60 kDa Ro SLE sera; lane 4—anti-La sera; lane 5—anti-52 kDa Ro sera; lane 6—anti-Sm/nRNP sera; lane 7— prestained protein molecular weight markers (10 Al).
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4. Discussion In an earlier report (Kurien and Scofield, 1998), it was shown that heat-mediated staining and destaining of SDS-PAGE gels could dramatically decrease staining and destaining time. Heat has been proposed to increase the permeability of the SDS-PAGE gel to the stain or the destain, thus helping to rapidly stain or destain the proteins on the gel. Also, it has been shown that heat can facilitate the elution of DNA from an agarose gel, when used in conjunction with pellet pestle homogenization (Kurien et al., 2001a,b). Here again, heat has been proposed to increase the permeability of the agarose gel, since homogenization in the absence of heat failed to elute any DNA. In the light of these experiments, we were interested to see whether heat could increase the permeability of the SDS-PAGE gels and thus enable the rapid electrophoretic transfer of proteins from the gel to nitrocellulose membranes. We have demonstrated that both high and low molecular weight proteins are quantitatively and rapidly transferred from SDS-PAGE gels to membranes by heat-mediated electro transfer, from gels of different acrylamide concentrations and thickness. The transfer was complete and there was no visible sign of any non-transferred residual protein, as evidenced by the transfer of the pre-stained protein molecular weight standards. The multiple minor bands seen in the 60 kDa Ro immunoblots are not an artifact of the transfer process, and are an inherent breakdown product of 60 kDa Ro, arising prior to the transfer process (Figs. 1 and 2). Methanol has been omitted from the transfer buffer owing to the fact that it may decrease the elution efficiency of large proteins by partially ‘‘fixing’’ them in the gel (Beisiegel, 1986; Bolt and Mahoney, 1997). Transferring high molecular weight proteins has been the bane of researchers everywhere. In our experience we have seen residual high molecular weight proteins in the gels with higher percentages of acrylamide (15%) even after a prolonged overnight transfer utilizing the conventional method. Prolonged electrophoresis lasting from 16 to 20 h in the presence of SDS has enabled the quantitative transfer of high molecular weight proteins (Otter et al., 1987). Using novel gel and blotting conditions Bolt and Mahoney, 1997, were able to efficiently transfer high molecular weight proteins.
The method detailed herein would prove useful for the rapid transfer of proteins, especially high molecular weight proteins, from SDS-PAGE gels to nitrocellulose membranes without the use of specialized equipment or chemicals. The fact that methanol is not required provides an added advantage.
Acknowledgements This work was supported by NIH grant ARO1844 and Oklahoma Center for the Advancement of Science and Technology to RHS. We also express our thanks to Samantha Ganick for her excellent technical assistance.
References Beisiegel, U., 1986. Review. Protein blotting. Electrophoresis 7, 1. Bolt, M.W., Mahoney, P.A., 1997. High efficiency blotting of proteins of diverse sizes following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Anal. Biochem. 247, 185. Deutscher, S.L., Harley, J.B., Keene, J.D., 1988. Molecular analysis of the 60 kD human Ro ribonucleoprotein. Proc. Natl. Acad. Sci. U. S. A. 85, 9479. Harley, J.B., Scofield, R.H., 1991. Systemic lupus erythematosus: RNA-protein autoantigens, models of disease heterogeneity, and theories of etiology. J. Clin. Immunol. 11, 297, Review. Kurien, B.T., Scofield, R.H., 1997. Multiple immunoblots after nonelectrophoretic bidirectional transfer of a single SDS-PAGE gel with multiple antigens. J. Immunol. Methods 205, 91. Kurien, B.T., Scofield, R.H., 1998. Heat mediated quick Coomassie blue protein staining and destaining of SDS-PAGE gels. Indian J. Biochem. Biophys. 35 (6), 385. Kurien, B.T., Newland, J., Paczkowski, C., Moore, K.L., Scofield, R.H., 2000. Association of neutropenia in systemic lupus erythematosus with anti-Ro and binding of an immunologically cross-reactive neutrophil membrane antigen. Clin. Exp. Immunol. 120, 209. Kurien, B.T., Kaufman, K.M., Harley, J.B., Scofield, R.H., 2001a. Pellet pestle homogenization of agarose gel slices at 45 jC for deoxyribonucleic acid extraction. Anal. Biochem. 296, 162. Kurien, B.T., Matsumoto, H., Scofield, R.H., 2001b. Purification of tryptic peptides for mass spectrometry using polyvinylidene fluoride membrane. Indian. J. Biochem. Biophys. 38, 274. Laemmli, U.K., 1970. Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227, 680. Otter, T., King, S.M., Witman, G.B., 1987. A two-step procedure for efficient electro-transfer of both high-molecular weight (greater than 400,000) and low-molecular weight (less than 20,000) proteins. Anal. Biochem. 162, 370. Peferoen, M., Huybrechts, R., De Loof, A., 1982. Vacuum-blotting: a new simple and efficient transfer of proteins from sodium
B.T. Kurien, R.H. Scofield / Journal of Immunological Methods 266 (2002) 127–133 dodecyl sulfate-polyacrylamide gels to nitrocellulose. FEBS Lett. 145, 369. Scofield, R.H., Farris, A.D., Horsfall, A.C., Harley, J.B., 1999. Fine specificity of the autoimmune response to the Ro/SSA and La/ SSB ribonucleoproteins. Arthritis Rheum. 42, 199, Review. Towbin, H., Staehelin, T., Gordon, J., 1979. Electrophoretic transfer
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of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. U. S. A. 76, 4350. Yamagata, H., Harley, J.B., Reichlin, M., 1984. Molecular properties of the Ro/SSA antigen and ELISA for quantitation of antibody. J. Clin. Invest. 74, 625.
Heat-mediated, ultra-rapid electrophoretic transfer of high and low molecular weight proteins to nitrocellulose membranes Biji T. Kurien a,*, R. Hal Scofield a,b,c a
Arthritis and Immunology Program, Oklahoma Medical Research Foundation, 825 NE 13th Street, Oklahoma City, OK 73104, USA b Department of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA c Department of Veterans Affairs Medical Center, Oklahoma City, OK 73104, USA Received 10 December 2001; received in revised form 8 April 2002; accepted 9 April 2002
Abstract Here, we report an ultra-rapid method for the transfer of high and low molecular weight proteins to nitrocellulose membranes following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In this procedure, the electrotransfer was performed with heated (70 – 75 jC) normal transfer buffer from which methanol had been omitted. Complete transfer of high and low molecular weight proteins (a purified protein, molecular weight protein standards and proteins from a human tissue extract) could be carried out in 10 min for a 0.75-mm, 7% SDS-PAGE gel. For 10% and 12.5% gels (0.75 mm), the corresponding time was 15 min. In the case of 1.5-mm gels, a complete transfer could be carried out in 20 min for 7%, 10% and 12.5% gels. The permeability of the gel is increased by heat, such that the proteins trapped in the polyacrylamide gel matrix can be easily transferred to the membrane. When the heat-mediated transfer method was compared with a conventional transfer protocol, under similar conditions, we found that the latter method transferred minimal low molecular weight proteins while retaining most of the high molecular weight proteins in the gel. In summary, this procedure is very rapid, avoids the use of methanol and is particularly useful for the transfer of high molecular weight proteins. D 2002 Elsevier Science B.V. All rights reserved. Keywords: SDS-PAGE; Western blotting; Nitrocellulose
1. Introduction Transfer of proteins from sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels to nitrocellulose or polyvinylidene difluoride membranes have been achieved in three different ways: (a) simple diffusion (Kurien and Scofield, 1997); vac*
Corresponding author. Tel.: +1-405-271-7394; fax: +1-405271-4110. E-mail address: [email protected] (B.T. Kurien).
uum-assisted solvent flow (Peferoen et al., 1982); and (c) electrophoretic elution (Towbin et al., 1979). There is considerable interest in diffusion-mediated transfer of proteins, since Kurien and Scofield (1997) showed that multiple immunoblots can be generated after nonelectrophoretic bidirectional transfer of a single SDSPAGE gel with multiple antigens. The lifts from SDSPAGE gels, for immunoblotting, using this method are particularly useful in identification of proteins by mass spectrometry (Kurien et al., 2000; Kurien et al., 2001a,b). However, electrophoretic elution, widely
0022-1759/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 2 - 1 7 5 9 ( 0 2 ) 0 0 1 0 3 - 5
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B.T. Kurien, R.H. Scofield / Journal of Immunological Methods 266 (2002) 127–133
used in most laboratories, is still the method of choice for transfer of proteins to membranes. Electrophoretic transfer of proteins to nitrocellulose, subsequent to separation by SDS-PAGE, is a fundamental step prior to detection of specific proteins with specific antibodies (Towbin et al., 1979). The Western transfer of proteins normally takes about 2 –4 h at about 70 V or an overnight transfer at 30 V. High molecular weight proteins are often stubbornly resistant to transfer (Bolt and Mahoney, 1997; Kurien and Scofield, unpublished observation) in spite of these prolonged runs. This problem is accentuated when higher percentage gels are used. Prolonged electrotransfer (16 – 20 h) at high current density coupled with inclusion of 0.01% sodium dodecyl sulfate, to enhance protein elution, has been used to efficiently transfer high-molecular weight proteins (Otter et al., 1987). Here, we show that both low and high molecular weight proteins can be very efficiently transferred to nitrocellulose membrane in a very short period of time using heated, normal transfer buffer without methanol.
The cells were lysed by sonication using a Branson sonicator using setting 4 in SDS-PAGE lysis buffer and spun at 10,000 g for 10 min. An aliquot of the supernatant was analyzed by SDS-PAGE, followed by immunoblotting. 2.2.2. SDS-PAGE SDS-PAGE was carried out according to Laemmli (1970). Bovine 60 kDa Ro (1 Ag/strip) and prestained protein standards (5 Al/lane) were separated on 7%, 10% or 12.5% gels (0.75- or 1.5mm thickness) and transferred to nitrocellulose. Protein standards were used at 10 Al/lane for the experiments that compared the efficiency of the heat transfer method with that obtained using a conventional method. 2.2.3. Electrophoretic transfer Conventional electro-transfer of proteins separated on gradient and regular gels was carried out at 4 jC using standard transfer buffer (25 mM Tris, 190 mM Glycine, 20% methanol) and compared with heatmediated electrophoretic transfer under similar conditions.
2. Materials and methods 2.1. Materials Nitrocellulose membranes were purchased from Gelman Sciences. Purified bovine 60 kDa Ro (Deutscher et al., 1988; Yamagata et al., 1984) was a gift from Immunovision (Springdale, AK). BenchMark pre-stained molecular weight standards were obtained from Gibco BRL (Bethesda, MD). All other chemicals used were of reagent grade. Western blot transfer apparatus, possessing the capability of circulating hot or cold water at the base, was purchased from Hoeffer Scientific Instruments (Pharmacia/Amersham). A refrigerator bath/circulating water bath (Endocal RT110), capable of circulating hot or cold water was from Neslab, Portsmouth, NH. Branson Sonifier Cell Disruptor 185 was from VWR Scientific, (Boston, MA). 2.2. Methods 2.2.1. Preparation of HeLa cell lysate Freshly cultured HeLa cells were harvested and washed twice with phosphate-buffered saline, pH 7.4.
2.2.4. Heat-mediated electrophoretic transfer The transfer was carried out in the absence of methanol, using 25 mM Tris and 190 mM glycine buffer. The buffer was heated to 70– 75 jC in a 1-l beaker that was covered with clear plastic wrap. The hot buffer was transferred to a Hoeffer transfer apparatus, fabricated with the capability of circulating hot water (70 –75 jC) at the base of the apparatus (the hot water being supplied by a re-circulating water bath which should be turned on at least 30 min before completion of the SDS PAGE procedure so as to allow the water to attain the required temperature). This step was carried out after the gel transfer sandwich had been assembled. To assemble the gel sandwich, the nitrocellulose was moistened with the same buffer, taking about 10 min to be moistened completely. (Alternately, the nitrocellulose membrane can be moistened almost instantaneously with regular transfer buffer that contains methanol.) Preferably, the buffer used to assemble the sandwich should be at room temperature or just slightly warm. (Using the hot buffer to assemble the sandwich causes the 3 M filter paper, used as a part of the gel sandwich, to disinte-
B.T. Kurien, R.H. Scofield / Journal of Immunological Methods 266 (2002) 127–133
grate partially). The cassette with the gel-membrane sandwich was placed in the transfer apparatus. The transfer apparatus was placed on a Corning PC-351 magnetic stirrer. A magnetic stir bar was used to circulate the buffer inside the transfer apparatus. The transfer was carried out at 40 V for periods ranging from 10 to 20 min, depending upon the gel used, using a FB300 power supply from Fisher Scientific (Houston, TX, USA). 2.2.5. Immunoblotting Standard immunoblotting was carried out (Towbin et al., 1979). Human systemic lupus erythematosus (SLE) sera with autoantibodies against 60 kDa Ro, 48 kDa La, 52 kDa Ro, Sm and nuclear ribonucleoprotein autoantigens (Harley and Scofield, 1991; Scofield et al., 1999) were used to identify the respective antigens transferred to nitrocellulose from various gels.
3. Results We have used purified protein, prestained molecular weight standards and a human cell extract to
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determine the efficiency of heat-mediated electroblotting. In order to determine the efficiency of heatmediated transfer of proteins, gels of two different thicknesses—0.75 and 1.5 mm—and gels with three different amounts of acrylamide (7%, 10% and 12.5%) were used in this study. In addition, transfer of proteins from a 4– 20% gradient SDS-PAGE gel and a 12.5% (1.5 mm) regular gel was also investigated and compared to that obtained using a conventional transfer method under similar conditions. Bovine 60 kDa Ro (lane 2 in Figs. 1 and 2) and prestained protein standards (Lane 4 in Figs. 1 and 2) could be transferred entirely with respect to gels with different thicknesses and polyacrylamide amounts. It took about 10 min to transfer the entire pre-stained protein marker (5 Al of the marker) from a 0.75 mm, 7% gel (Fig. 1 A, lane 4). All the protein markers, ranging from 184 to 9 kDa, could be transferred to membranes from 7%, 10% and 12.5% gels (Fig. 1B,C, lane 4) in 15 min. The post-transfer polyacrylamide gels were clean, without any sign of residual non-transferred protein markers. It took 20 min to transfer all the protein markers in the case of the 1.5-mm gels (7%, 10% and 12.5% gels) (Fig. 2, lane 4).
Fig. 1. Western blot transfer and immunoblotting of bovine 60 kDA Ro and prestained molecular weight standards using 7%, 10% and 12.5% gels (0.75 mm gels). (A, B, C) Proteins on a 7%, 10% and 12.5% SDS-PAGE gels respectively transferred as mentioned in Materials and methods. Lane 1—anti-60 kDa negative control; lane 2—anti-Ro positive control; lane 3—conjugate control; lane 4—prestained molecular weight standards.
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Fig. 2. Western blot transfer and immunoblotting of bovine 60 kDa Ro and prestained molecular weight standards using 7%, 10% and 12.5% gels (1.5 mm gels). (A, B, C) Proteins on a 7%, 10% and 12.5% SDS-PAGE gels transferred as mentioned in Materials and methods. Lane 1—anti-60 kDa negative control; lane 2—anti-Ro positive control; lane 3—conjugate control; lane 4—prestained molecular weight standards.
To compare the efficiency of the transfer process using this method to that obtained with a conventional method, we carried out two experiments. In the first experiment, we analyzed pre-stained protein ladder and purified 60 kDa Ro on a 12.5% SDS-PAGE gel. One half of the gel, containing the marker (10 Al) and 60 kDa Ro (4 Ag/well), was transferred using our method. The other half, containing equal amount of the marker (10 Al) and 60 kDa Ro (4 Ag/well) was transferred, using a conventional transfer procedure as mentioned in Materials and methods. Similarly, equal volumes of marker and HeLa cell extract were analyzed on a 4 – 20% pre-cast gradient gel. One half of the gel, containing the HeLa cell extract and the protein marker, was transferred using our method and the other half, containing the same amount of the extract and marker, with a conventional method. Fig. 3 shows the Fast Green stained nitrocellulose membranes obtained after transfer of protein using our method (right) and a conventional method (left) from a 4 – 20% gradient gel. The transfer of the prestained molecular weight standards and the proteins from the lysate was very efficient with the heat transfer method compared to the conventional method. The low molecular weight markers and low
molecular weight proteins in the lysate have been barely transferred after 20 min of conventional transfer, while higher molecular weight proteins have not been transferred. Fig. 4A shows purified 60 kDa Ro and the markers transferred with the new heat method (left) compared to that obtained with the conventional method (right) using a 12.5% gel. An anti-60 kDa Ro containing lupus sera binds strongly to 60 kDa Ro in the blot on the left, much stronger than it does on the right. In addition, molecular weight markers (lane 5, left) have been transferred efficiently, while only the lower molecular weight proteins were just barely transferred (lane 5, right). This clearly shows that the transfer was more efficient using the heat transfer compared to the regular transfer. Fig. 4B shows the immunoblot of HeLa extract obtained after transfer, from a 4– 20% gradient gel, using our method (left) and that obtained using a conventional method (right). Both the gels were transferred under similar conditions. Anti-60 kDa Ro, antiLa, anti-52 kDa Ro and anti-Sm/nRNP containing lupus sera (lanes 3 – 6, respectively; left) bind very strongly to their respective antigens on the blot obtained using the heat transfer method. Whereas
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could just barely be transferred using the conventional method (lane 7, right). All the markers could be efficiently transferred using the heat transfer procedure (lane 7, left). This, once again, shows that the transfer of proteins is partial after a 20-min transfer using a conventional means of transferring protein from a gel to the membrane.
Fig. 3. Fast Green staining of proteins from a HeLa cell extract transferred to nitrocellulose membrane by a conventional method (left) and using the heat transfer method (right) from a 4 – 20% SDSPAGE gradient gel. Lane 1—HeLa cell extract; lane 2—10 Al of prestained molecular weight marker.
only the anti-Ro sera and anti-Sm/nRNP sera bind, in the case of the blot that had been transferred using a conventional method, and the binding is weak (lanes 3 and 6, right). In addition, only the low molecular weight protein markers (16.8, 21.7 and 28.4 kDa)
Fig. 4. Immunoblots of purified 60 kDa Ro and proteins derived from a HeLa cell extract transferred to nitrocellulose membrane using the heat transfer method (left) and by a conventional method (right). (A) Purified 60 kDa Ro autoantigen immunoblot obtained from a 12.5% SDS-PAGE gel using the heat transfer method (left) and by a conventional method (right). Lane 1—conjugate control; lanes 2 and 3—normal controls; lane 4—anti-60 kDa Ro SLE sera; lane 5—prestained protein molecular weight standards. (B) HeLa cell extract immunoblot obtained from a 4 – 20% gradient SDSPAGE gel using the heat transfer method (left) and a conventional transfer method (right). Lane1—conjugate control; lane 2—normal control; lane 3—anti-60 kDa Ro SLE sera; lane 4—anti-La sera; lane 5—anti-52 kDa Ro sera; lane 6—anti-Sm/nRNP sera; lane 7— prestained protein molecular weight markers (10 Al).
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4. Discussion In an earlier report (Kurien and Scofield, 1998), it was shown that heat-mediated staining and destaining of SDS-PAGE gels could dramatically decrease staining and destaining time. Heat has been proposed to increase the permeability of the SDS-PAGE gel to the stain or the destain, thus helping to rapidly stain or destain the proteins on the gel. Also, it has been shown that heat can facilitate the elution of DNA from an agarose gel, when used in conjunction with pellet pestle homogenization (Kurien et al., 2001a,b). Here again, heat has been proposed to increase the permeability of the agarose gel, since homogenization in the absence of heat failed to elute any DNA. In the light of these experiments, we were interested to see whether heat could increase the permeability of the SDS-PAGE gels and thus enable the rapid electrophoretic transfer of proteins from the gel to nitrocellulose membranes. We have demonstrated that both high and low molecular weight proteins are quantitatively and rapidly transferred from SDS-PAGE gels to membranes by heat-mediated electro transfer, from gels of different acrylamide concentrations and thickness. The transfer was complete and there was no visible sign of any non-transferred residual protein, as evidenced by the transfer of the pre-stained protein molecular weight standards. The multiple minor bands seen in the 60 kDa Ro immunoblots are not an artifact of the transfer process, and are an inherent breakdown product of 60 kDa Ro, arising prior to the transfer process (Figs. 1 and 2). Methanol has been omitted from the transfer buffer owing to the fact that it may decrease the elution efficiency of large proteins by partially ‘‘fixing’’ them in the gel (Beisiegel, 1986; Bolt and Mahoney, 1997). Transferring high molecular weight proteins has been the bane of researchers everywhere. In our experience we have seen residual high molecular weight proteins in the gels with higher percentages of acrylamide (15%) even after a prolonged overnight transfer utilizing the conventional method. Prolonged electrophoresis lasting from 16 to 20 h in the presence of SDS has enabled the quantitative transfer of high molecular weight proteins (Otter et al., 1987). Using novel gel and blotting conditions Bolt and Mahoney, 1997, were able to efficiently transfer high molecular weight proteins.
The method detailed herein would prove useful for the rapid transfer of proteins, especially high molecular weight proteins, from SDS-PAGE gels to nitrocellulose membranes without the use of specialized equipment or chemicals. The fact that methanol is not required provides an added advantage.
Acknowledgements This work was supported by NIH grant ARO1844 and Oklahoma Center for the Advancement of Science and Technology to RHS. We also express our thanks to Samantha Ganick for her excellent technical assistance.
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