西方點墨法(western blot)在分子生物學、生物化學和分子免疫學常用的一種實驗方法，可以分析蛋白質表現及蛋白質重量。蛋白質標誌(Protein marker)在西方點墨法中扮演的角色是做為測定蛋白質大小的標準。但是這些蛋白質標記還是有缺點存在，例如這些蛋白質標記無法自動呈色，必須利用手工方式來繪上位置，如此一來會增加誤差的機率。此外，市售蛋白質標記的分子量大小並無規律性，無法利用不同大小標記之間的距離來推估分子量。經過預染的蛋白質標記會因為電性的改變而產生分子量誤差，會較預期的分子量來的更大。

Accurate determination of protein molecular weights is important for Western blot analysis(Towbin et al., 1979). Protein marker plays a very important role in the Western blot, it is composed of different molecular weight proteins to present bands on SDS PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and western blot. According to different molecule weight band of the protein marker, investigators can estimate the molecule weight of protein sample and figure out if the sample is correct or not. In these experiments, investigators usually rely on pre-stained protein marker to analyze the protein sample(Parkinson and Redshaw, 1984) (Tsang et al., 1984). The advantages of the pre-stained protein marker is the band is stained with different color dye which can be clearly observed when we run the SDS PAGE gel.

論文審定書 i
Acknowledgement ii
Chinese Abstract iii
Abstract iv
Contents v
List of Figures vi
List of Tables vii
Abbreviations viii
Introduction 1
Material and methods 6
Results 13
Discussion 17
Figures 21
Tables 34
References 38

Alix, A.J. (1999). Predictive estimation of protein linear epitopes by using the program PEOPLE. Vaccine 18, 311-314.

Barlow, D.J., Edwards, M.S., and Thornton, J.M. (1986). Continuous and discontinuous protein antigenic determinants. Nature 322, 747-748.

Bratthauer, G.L. (1999). The avidin-biotin complex (ABC) method and other avidin-biotin binding methods. Methods in molecular biology (Clifton, NJ) 115, 203-214.

Bulashevska, A., and Eils, R. (2006). Predicting protein subcellular locations using hierarchical ensemble of Bayesian classifiers based on Markov chains. BMC bioinformatics 7, 298.

Burgess, R.R., Arthur, T.M., and Pietz, B.C. (2000). Mapping protein-protein interaction domains using ordered fragment ladder far-western analysis of hexahistidine-tagged fusion proteins. Methods in enzymology 328, 141-157.

Chang, M., Hsu, H.Y., and Lee, H.J. (2005). Dye-free protein molecular weight markers. Electrophoresis 26, 3062-3068.

Chen, J., Liu, H., Yang, J., and Chou, K.C. (2007). Prediction of linear B-cell epitopes using amino acid pair antigenicity scale. Amino acids 33, 423-428.

Compton, M.M., Lapp, S.A., and Pedemonte, R. (2002). Generation of multicolored, prestained molecular weight markers for gel electrophoresis. Electrophoresis 23, 3262-3265.

Daskalow, K., Boisguerin, P., Jandrig, B., Volkmer, R., Micheel, B., and Schenk, J.A. (2008). Epitope mapping of antibodies against S-tagged fusion proteins and molecular weight markers. Bioscience, biotechnology, and biochemistry 72, 346-351.

Della-Penna, D., Christoffersen, R.E., and Bennett, A.B. (1986). Biotinylated proteins as molecular weight standards on Western blots. Analytical biochemistry 152, 329-332.


El-Manzalawy, Y., Dobbs, D., and Honavar, V. (2008). Predicting linear B-cell epitopes using string kernels. J Mol Recognit 21, 243-255.

Elia, G. (2008). Biotinylation reagents for the study of cell surface proteins. Proteomics 8, 4012-4024.

Fox, J.D., Kapust, R.B., and Waugh, D.S. (2001). Single amino acid substitutions on the surface of Escherichia coli maltose-binding protein can have a profound impact on the solubility of fusion proteins. Protein science : a publication of the Protein Society 10, 622-630.

Gabaldon, T., and Huynen, M.A. (2004). Prediction of protein function and pathways in the genome era. Cellular and molecular life sciences : CMLS 61, 930-944.

Huang, J., Honda, W., and Kanehisa, M. (2007). Predicting B cell epitope residues with network topology based amino acid indices. Genome informatics International Conference on Genome Informatics 19, 40-49.


Kao, C.H., Cheng, C.M., Chuang, K.H., Chuang, C.H., Tzou, S.C., Cheng, T.C., Hsieh, Y.C., Liao, K.W., Wang, Y.M., Chang, L.S., et al. (2012). A regularly spaced and self-revealing protein ladder for anti-tag Western blot analysis. Analytical biochemistry 431, 1-3.

Larsen, J.E., Lund, O., and Nielsen, M. (2006). Improved method for predicting linear B-cell epitopes. Immunome research 2, 2.

Lindbladh, C., Mosbach, K., and Bulow, L. (1991). Standard calibration proteins for Western blotting obtained by genetically prepared protein A conjugates. Analytical biochemistry 197, 187-190.

Luo, W., Liang, T.C., Li, J.M., Hsieh, J.T., and Lin, S.H. (1996). A universal tag for recombinant proteins. Archives of biochemistry and biophysics 329, 215-220.

Nakajima, K., and Yaoita, Y. (1997). Construction of multiple-epitope tag sequence by PCR for sensitive Western blot analysis. Nucleic acids research 25, 2231-2232.


Neumaier, M., Fenger, U., and Wagener, C. (1986). Transblot studies with biotin-labeled proteins: electrophoretic mobilities and detection limits. Analytical biochemistry 156, 76-80.

Parkinson, D., and Redshaw, J.D. (1984). Visible labeling of proteins for polyacrylamide gel electrophoresis with dabsyl chloride. Analytical biochemistry 141, 121-126.

Rydzanicz, R., Zhao, X.S., and Johnson, P.E. (2005). Assembly PCR oligo maker: a tool for designing oligodeoxynucleotides for constructing long DNA molecules for RNA production. Nucleic acids research 33, W521-525.

Saha, S., and Raghava, G.P. (2006). Prediction of continuous B-cell epitopes in an antigen using recurrent neural network. Proteins 65, 40-48.

Smyth, D.R., Mrozkiewicz, M.K., McGrath, W.J., Listwan, P., and Kobe, B. (2003). Crystal structures of fusion proteins with large-affinity tags. Protein science : a publication of the Protein Society 12, 1313-1322.

Sollner, J., Grohmann, R., Rapberger, R., Perco, P., Lukas, A., and Mayer, B. (2008). Analysis and prediction of protective continuous B-cell epitopes on pathogen proteins. Immunome research 4, 1.

Sorensen, H.P., and Mortensen, K.K. (2005). Soluble expression of recombinant proteins in the cytoplasm of Escherichia coli. Microbial cell factories 4, 1.

Terpe, K. (2003). Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems. Applied microbiology and biotechnology 60, 523-533.

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 the United States of America 76, 4350-4354.

Tsang, V.C., Hancock, K., and Simons, A.R. (1984). Calibration of prestained protein molecular weight standards for use in the "Western" or enzyme-linked immunoelectrotransfer blot techniques. Analytical biochemistry 143, 304-307.
Yu, U., Lee, S.H., Kim, Y.J., and Kim, S. (2004). Bioinformatics in the post-genome era. Journal of biochemistry and molecular biology 37, 75-82.

Zhang, Y.M., Geng, L., and Huang, D.W. (2009). Generate Western blot protein marker from a single construct. Analytical biochemistry 390, 206-208.