Voltage profile generation for simultaneous multi-protein detection in western blot analysis

Matthew Blair, Mina Wanis, Gaurav Swarnkar, Hiroki Yokota, Stanley Chien

Department of Biomedical Engineering, Indiana University-Purdue University Indianapolis, Indianapolis, USA.
Department of Electrical and Computer Engineering, Indiana University-Purdue University Indianapolis, Indianapolis, USA.

DOI: 10.4236/jbise.2012.59067                

Abstract

Western blotting is a popular technique for examining expression levels of proteins using gel-based electrophoretic fractionation followed by blotting and antibody reactions. Although this is a mature technique, one of the major limitations is the need to prepare an individual electrophoretic gel for each of the protein species to be analyzed. Since most analyses require the detection of multiple protein species, a procedure that allows utilization of a single gel for detecting multiple protein species should significantly save time and resources. In this paper, we developed a novel multiprotein detection device, which enabled simultaneous detection of several proteins species from a single electrophoretic gel. In this device, a protein transfer unit utilized a multi-anode plate that generated a non-uniform voltage profile. This voltage profile enabled uniform transfer regardless of molecular mass of proteins. In vitro experiments using samples, isolated from boneforming osteoblast cells, showed that the expression levels of 5 - 7 different proteins were detectable in the presence and absence of mechanical stimulation that activated genes necessary for bone formation. The result supports the notion that through simultaneous detection of multiple protein species, the described device contributes to reduction in procedural time and sample amounts, as well as a removal of variations among multiple gels.

Keywords

Western Blot; Protein Transfer; Voltage Control; Multiple Membranes

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1]	Hagyousif, A.M., Chong, V.J., Yokota, H. and Chien, S. (2010) Development of a novel protein multi-blotting device. Journal of Biomedical Science and Engineering, 3, 1125-1132. doi:10.4236/jbise.2010.312146
[2]	Towbin, H., Staehelin, T. and Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proceedings of the National Academy of Sciences of USA, 76, 4350-4354. doi:10.1073/pnas.76.9.4350
[3]	Burnette, W.N. (1981) “Western blotting”: Electrophoretic transfer of proteins from sodium dodecyl sulfatepolyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein. Analytical Biochemistry, 112, 195-203. doi:10.1016/0003-2697(81)90281-5
[4]	Chen, H.-W. (2003) Semidry electroblotter. Wealtec Enterprise Co. Ltd., San Francisco, 1-7.
[5]	Szewczyk, B. and Kozloff, L.M. (2004) A method for the efficient blotting of strongly basic proteins from sodium dodecyl sulfate-polyacrylamide gels to nitrocellulose. Analytical Biochemistry, 150, 403-407. doi:10.1016/0003-2697(85)90528-7
[6]	Avrameas, S. and Guilbert, B. (1971) A method for quantitative determination of cellular immunoglobulins by enzyme-labeled antibodies. European Journal of Immunology, 1, 394-396. doi:10.1002/eji.1830010518
[7]	Kuhlmann, W.D., Avrameas, S. and Ternynck, T. (1974) A comparative study for ultrastructural localization of intracellular immunoglobulins using peroxidase conjugates. Journal of Immunological Methods, 5, 33-48. doi:10.1016/0022-1759(74)90043-X
[8]	Mathews, S.T., Plaisance, E.P. and Kim, T. (2009) Imaging systems for westerns: chemiluminescence vs. infrared detection. Protein Blotting and Detection, 536, 499-513. doi:10.1007/978-1-59745-542-8_51
[9]	Hirasawa, H., Jiang, C., Zhang, P., Yang, F., Yokota, H. (2010) Mechanical stimulation suppresses phosphorylation of eIF2alpha and PERK-mediated responses to stress to the endoplasmic reticulum. FEBS Letters, 584, 745-752. doi:10.1016/j.febslet.2009.12.028
[10]	Wang, D., et al. (1999) Isolation and characterization of MC3T3-E1 preosteoblast subclones with distinct in vitro and in vivo differentiation/mineralization potential. Journal of Bone and Mineral Research, 14, 893-903. doi:10.1359/jbmr.1999.14.6.893
[11]	Giorgos Lazaridis, “PWM Modulation”. http://pcbheaven.com/wikipages/PWM_Modulation/
[12]	Hamamura, K., Swarnkar, G., Tanjung, N., Cho, E., Li, J., Na, S. and Yokota, H. (2012) RhoA-mediated signaling in mechanotransduction of osteoblasts. Connective Tissue Research, 53, 398-406. doi:10.3109/03008207.2012.671398