Summary
Isoelectric focusing as used in the first dimension of two-dimensional gel electrophoresis separates protein isoforms such as those due to phosphorylation and acetylation. The immunoblotting method described here reveals this diversity by a one-dimensional separation. Using commercially available immobilized pH gradient plates or strips, the resolved proteins are transferred to PVDF membranes by diffusion and are probed with protein specific antibody. The system is useful for monitoring changes of banding patterns and permits parallel processing of samples. Since the effect of posttranslational modifications on the isoelectric point can be predicted, inferring the number and extent of modifications is possible.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Towbin, H., Ozbey, O., and Zingel, O. (2001) An immunoblotting method for high-resolution isoelectric focusing of protein isoforms on immo-bilized pH gradients. Electrophoresis 22, 1887–1893.
Görg, A., Weiss, W., and Dunn, M.J. (2004) Current two-dimensional electrophoresis techno-logy for proteomics. Proteomics 4, 3665–3685.
Hoving, S., Voshol, H., and van Oostrum, J. (2005) Using Ultra-Zoom Gels for High-Resolution Two-Dimensional Polyacrylamide Gel Electrophoresis, The Proteomics Protocols Handbook, Humana Press Inc.,U.S., John M. Walker, ed. pp. 151–166
Rabilloud, T. (1998) Use of thiourea to increase the solubility of membrane proteins in two-dimensional electrophoresis. Electrophoresis 19, 758–760.
Bradford, M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72, 248–254.
Bjellqvist, B., Hughes, G.J., Pasquali, Ch., Paquet, N., Ravier, F., Sanchez, J.-Ch., et al (1993) The focusing positions of polypeptides in immobilized pH gradients can be predicted from their amino acid sequences. Electrophoresis 14, 1023–1031.
Obenauer, J.C., Cantley, L.C., and Yaffe, M.B. (2003) Scansite 2.0: Proteome-wide prediction of cell signaling interactions using short sequence motifs. Nucleic Acids Res. 31, 3635–3641.
Towbin, H., Bair, K.W., DeCaprio, J.A., Eck, M.J., Kim, S., Kinder, F.R., et al (2003) Proteomics-based target identification: bengamides as a new class of methionine aminopeptidase inhibitors. J Biol Chem. 278, 52964–52971.
Poland, J., Bohme, A., Schubert, K., and Sinha, P. (2002) Revisiting electroblotting of immobilized pH gradient gels: a new protocol for studying post-translational modification of proteins. Electrophoresis 23, 4067–4071.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2009 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Towbin, H. (2009). Blotting from Immobilized pH Gradient Gels: Application to Total Cell Lysates. In: Kurien, B., Scofield, R. (eds) Protein Blotting and Detection. Methods in Molecular Biology, vol 536. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-59745-542-8_27
Download citation
DOI: https://doi.org/10.1007/978-1-59745-542-8_27
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-934115-73-2
Online ISBN: 978-1-59745-542-8
eBook Packages: Springer Protocols