Abstract
Denaturing, discontinuous electrophoresis in the presence of SDS has become a standard method for the protein scientist. However, there are situations where this method produces suboptimal results. In these cases electrophoresis in the presence of positively charged detergents like cetyltrimethylammonium bromide (CTAB) may work considerably better. Methods for electrophoresis, staining, and blotting of such gels are presented.
Key words
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Laemmli U (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A 76:4350–4354
Dunn S (1986) Effects of the modification of transfer buffer composition and the renaturation of proteins in gels on the recognition of proteins on western blots by monoclonal antibodies. Anal Biochem 157:144–153
Shirahama K, Tsujii K, Takagi T (1974) Free boundary electrophoresis of sodium dodecyl sulphate-protein polypeptide complexes with special reference to SDS polyacrylamide gel electrophoresis. J Biochem 75:309–319
Rabilloud T, Girardot V, Lawrence J-J (1996) One-and two dimensional histone separations in acidic gels: usefulness of methylene blue-driven photo-polymerisation. Electrophoresis 17:67–73
Panyim S, Thitipongpanich R, Supatimusro D (1977) A simplified gel electrophoretic system and its validity for molecular weight determinations of protein cetyltrimethylammonium complexes. Anal Biochem 81:320–327
Eley M, Burns P, Kannapell C, Campbell P (1979) Cetyltrimethylammonium bromide polyacrylamide gel electrophoresis: estimation of protein subunit molecular weights using cationic detergents. Anal Biochem 92:411–419
MacFarlane D (1983) Use of benzyldimethyl-n-hexadecylammonium chloride (“16-BAC”), a cationic detergent, in an acidic polyacrylamide gel electrophoresis system to detect base labile protein methylation in intact cells. Anal Biochem 132:231–235
Mócz G, Bálint M (1984) Use of cationic detergents for polyacrylamide gel electrophoresis in multiphasic buffer systems. Anal Biochem 143:283–292
Akin D, Shapira R, Kinkade J Jr (1985) The determination of molecular weights of biologically active proteins by cetyltrimethylammonium bromide polyacrylamide gel electrophoresis. Anal Biochem 145:170–176
Akins R, Levin P, Tuan R (1992) Cetyltrimethylammonium bromide discontinuous electrophoresis: Mr-based separation of proteins with retention of enzymatic activity. Anal Biochem 202:172–178
Hartinger J, Stenius K, Högemann D, Jahn R (1996) 16-BAC/SDS-PAGE: a two-dimensional gel electrophoresis system suitable for the separation of integral membrane proteins. Anal Biochem 240:126–133
Ornstein L (1962) Disk electrophoresis: I. Background and theory. Ann N Y Acad Sci 121:321–351
Jovin T (1973) Multiphasic zone electrophoresis. I. Steady-state moving-boundary systems formed by different electrolyte combinations. Biochemistry 12:871–879
Jovin T (1973) Multiphasic zone electrophoresis. II. Design of integrated discontinuous buffer systems for analytical and preparative fractionation. Biochemistry 12:879–890
Jovin T (1973) Multiphasic zone electrophoresis. III. Further analysis and new forms of discontinuous buffer systems. Biochemistry 12:890–898
Jovin T (1973) Multiphasic zone electrophoresis. IV. Design and analysis of discontinuous buffer systems with a digital computer. Ann N Y Acad Sci 209:477–496
Chrambach A (1985) The practice of quantitative gel electrophoresis. VCH, Weinheim
Buxbaum E (2003) Cationic electrophoresis and electrotransfer of membrane glycoproteins. Anal Biochem 314:70–76
Pritchard D, Crawford C, Duce I, Behnke J (1985) Antigen stripping from the nematode epicuticle using the cationic detergent cetyltrimethylammonium bromide (CTAB). Parasite Immunol 7:575–585
Freedman D, Nutman T, Ottesen E (1988) Enhanced solubilization of immunoreactive proteins from Brugia malayi adult parasites using cetyltrimethylammonium bromide. Experim Parasitol 65:244–250
Maki K, Sagara J, Kawai A (1991) A cationic detergent, cetyltrimethylammonium bromide (CTAB), selectively dissociates the intermediate filament of the fibroblast. Biochem Biophys Res Commun 175:768–774
Smith I, Cromie R, Stainsby K (1988) Seeing gel wells well. Anal Biochem 169:370–371
Lazar J, Taub F (1992) A highly sensitive method for detecting peroxidase in situ hybridization or immunohistochemical assays. In: Kessler C (ed) Nonradioactive labeling and detection of biomolecules. Springer, Berlin, pp 135–142
Leong M, Fox G (1990) Luminescent detection of immunodot and Western blot. Meth Enzymol 184:442–451
Rodbard D, Chrambach A (1971) Estimation of molecular radius, free mobility, and valence using polyacrylamide gel electrophoresis. Anal Biochem 40:95–134
Stellwagen N (1998) Apparent pore size of polyacrylamide gels. Electrophoresis 19:1542–1547
Kramer ML (2006) A new multiphasic buffer system for benzyldimethyl-n-hexadecylammonium chloride polyacrylamide gel electrophoresis of proteins providing efficient stacking. Electrophoresis 27(2):347–356 doi:10.1002/elps.200500563
Heukeshoven J, Dernick R (1988) Improved silver staining procedure for fast staining in phastsystem development unit I. Staining of sodium dodecyl sulfate gels. Electrophoresis 9:28–32
Merril C, Goldman D, Sedman S, Ebert M (1981) Ultrasensitive stain for proteins in polyacrylamide gels shows regional variations in cerebrospinal fluid proteins. Science 211:1437–1438
Segrest J, Jackson R (1972) Molecular weight determination of glycoproteins by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Meth Enzymol 28:54–63
Thornton D, Holmes D, Sheehan J, Carlstedt I (1989) Quantitation of mucus glycoproteins blotted onto nitrocellulose membranes. Anal Biochem 182:160–164
Hancock K, Tsang V (1983) India ink staining of proteins on nitrocellulose paper. Anal Biochem 133:157–162
Hassan J, Feighery C, Whalan A (1987) Staining molecular weight markers on nitrocellulose using Ponceau S. J Clin Lab Immunol 24:104
Moore M, Viselli S (2000) Staining and quantification of proteins transferred to polyvinylidene difluoride membranes. Anal Biochem 279:241–242
Rabilloud T, Strub J-M, Luche S, Dorsselaer AV, Lunardi J (2001) A comparison between Sypro ruby and rubidium (II) tris(bathophenanthroline disulfonate) as fluorescent stains for protein detection in gels. Proteomics 1:699–704
Buxbaum E (2012) Fluorescent staining of gels. Meth Mol Biol 869:5
Acknowledgement
This work was supported in part by Kuwait University grant MPB029.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2015 Springer Science+Business Media New York
About this protocol
Cite this protocol
Buxbaum, E. (2015). Cationic Electrophoresis and Eastern Blotting. In: Kurien, B., Scofield, R. (eds) Western Blotting. Methods in Molecular Biology, vol 1312. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2694-7_19
Download citation
DOI: https://doi.org/10.1007/978-1-4939-2694-7_19
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-2693-0
Online ISBN: 978-1-4939-2694-7
eBook Packages: Springer Protocols