COMMENTARY
Problems with Multiple Use of Transfer Buffer in Protein Electrophoretic Transfer Yaser Dorri,1,2,* Biji T. Kurien,1 and R. Hal Scofield1,2,3 1
Arthritis and Immunology Program, Oklahoma Medical Research Foundation, 2Department of Medicine, University of Oklahoma Health Science Center, and 3Department of Veterans Affairs Medical Center, Oklahoma City, Oklahoma 73104, USA Two-dimensional gel electrophoresis (2DE) and SDS-PAGE are the two most useful methods in protein separation. Proteins separated by 2DE or SDS-PAGE are usually transferred to membranes using a variety of methods, such as electrophoretic transfer, heat-mediated transfer, or nonelectrophoretic transfer, for specific protein detection and/or analysis. In a recent study, Pettegrew et al.1 claim to reuse transfer buffer containing methanol for at least five times for transferring proteins from SDS-PAGE to polyvinylidene difluoride. They add 150 –200 ml fresh transfer solution each time for extended use as a result of loss of transfer buffer. Finally, they test efficiency of each protein transfer by chemiluminescence detection. Here, we comment on this report, as we believe this method is not accurate and useful for protein analysis, and it can cause background binding as well as inaccurate protein analysis. KEY WORDS: SDS-PAGE, Western blotting, immunoblotting, two-dimensional gel electrophoresis, transfer buffer, 2DE
INTRODUCTION 2
Two-dimensional gel electrophoresis (2DE) and SDSPAGE3 are the most useful methods in protein separation. Proteins separated by 2DE or SDS-PAGE are then transferred to membranes, using a variety of methods, such as electrophoretic transfer,4 heat-mediated transfer,5 or nonelectrophoretic transfer,6 for specific protein detection and/or analysis. Specific proteins can be detected using chemiluminescence method.7 In a recent study, Pettegrew et al.1 claim to reuse transfer buffer containing methanol at least five times transferring proteins from SDS-PAGE to polyvinylidene difluoride (PVDF membrane). It is mentioned that they add 150 –200 ml fresh transfer solution each time for extended use as a result of loss of transfer buffer. Finally, they test efficiency of each protein transfer by chemiluminescence detection. Here, we comment on this report, as we believe this method is neither accurate nor useful for protein analysis and believe it can cause background as well as inaccurate protein analysis. First of all, losing 200 ml buffer for every transfer is not normal. This means for every electrophoresis transfer, they use approximately 20% of fresh buffer; as a result, for every *ADDRESS CORRESPONDENCE TO: Yaser Dorri, Arthritis and Immunology, MS No. 24, 825 NE 13th St., Oklahoma City, OK 73104, USA (Phone: (405) 271-7394; E-mail:
[email protected] or
[email protected])
Journal of Biomolecular Techniques 21:1–2 © 2010 ABRF
five electrophoretic transfers, they have refreshed transfer buffer completely. It is understandable to lose approximately 50 ml transfer buffer for each transfer. However, it is difficult to understand the loss of 200 ml transfer solution after each transfer. The most transfer buffer that we lose after an electrophoretic transfer is 40 –50 ml. Secondly, it is possible that methanol could be lost as a result of evaporation after each transfer. The presence of methanol, which was introduced originally by Harry Towbin (Swiss Federal Institute of Technology, Zurich, Switzerland), helps to remove SDS from proteins as well as help to bind proteins to the membrane.8 Therefore, absence or evaporation of methanol level in transfer buffer can reduce the quality of protein transfer significantly. Finally, it is important to mention that proteins in the gel, prior to electrophoretic transfer, are not fixed. Therefore, some proteins can diffuse into the buffer. Also, for every transfer, there is a possibility of blowthrough, where proteins, especially low molecular weight proteins, pass through the membrane. These proteins, in every additional electrophoretic transfer, will bind nonspecifically to the next membrane. The least problem that these free proteins can cause is background binding, meaning that for every additional transfer, more background is observed while detecting proteins. Moreover, transferred proteins to the PVDF membrane after the second, third, fourth, or fifth electrophoretic transfer will not be good at all for protein
Y. DORRI ET AL. / PROBLEMS WITH MULTIPLE USE OF TRANSFER BUFFER
every use. This is very important for clinical protein analysis.
analysis, as the mixture of proteins binds to membrane and possibly to the proteins of interest. Even in Figure 1, section A, of the article published by Pettegrew et al.,1 a shift of the protein marker is seen easily on the second, third, and consequent transfers. Specifically, for the bottom three protein markers, the shift is very significant. This is perhaps caused by protein modification, evaporation of methanol in the buffer, and/or poor buffering capacity. It is known that buffering capacity is reduced after the second transfer, causing high molecular weight proteins not to transfer well.9 This is an obvious transfer problem and is observed in Figure 1, section A, as high molecular weight proteins are not transferred well in second, third, and consequent transfers. There is also a large variation in the quality of protein transfers in Figure 1, section A. Also, in Figure 1, section B, binding of other proteins is observed above and below the specified protein band (5⫻ transfer). Background is a major concern with this method and is observed easily for additional electrophoretic transfer in Figure 1, section A. Therefore, we do not recommend multiple electrophoretic transfers of proteins for more than one time, specifically if the transferred proteins are different in
REFERENCES 1. Pettegrew CJ, Jayini R, Islam MR. Transfer buffer containing methanol can be reused multiple times in protein electrotransfer. J Biomol Tech 2009;20:93–95. 2. Dorri Y, Kurien BT, Scofield RH. A simpler and faster version of two-dimensional gel electrophoresis using vertical, mini SDSPAGE apparatus. Iran J Chem Chem Eng 2009;28:51–56. 3. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227:680. 4. Kurien BT, Scofield RH. Western blotting. Methods 2006;8:283– 293. 5. Kurien BT, Scofield RH. Heat-mediated, ultra-rapid electrophoretic transfer of high and low molecular weight proteins to nitrocellulose membranes. J Immunol Methods 2002;266:127– 133. 6. Kurien BT, Scofield RH. Multiple immunoblots after non-electrophoretic bidirectional transfer of a single SDS-PAGE gel with multiple antigens. J Immunol Methods 1997;205:94 –97. 7. Dorri Y, Khalili AW, Scofield RH. Whole and strip nitrocellulose membrane as well as a new-line-immunoblotting of antigen using the chemiluminescence technique. Methods Mol Biol 2009;536: 417– 432. 8. Kurien BT, Scofield RH. Introduction to protein blotting. Methods Mol Biol 2009;536:9 –22. 9. Grafin, DE. Cell Structure, A Practical Approach. Oxford University Press, 2003; Volume 1: 197–268.
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JOURNAL OF BIOMOLECULAR TECHNIQUES, VOLUME 21, ISSUE 1, APRIL 2010
