Urea Gradient Gel Electrophoresis
Urea gradient gel electrophoresis is a simple method for monitoring the denaturant-induced unfolding of proteins. The method was first introduced for this purpose by T.E. Creighton in 1979. Urea gradient gels are slab gels prepared with a horizontal gradient of urea concentration, usually 0 - 8 M. A single sample of protein is applied across the top of the gel and electrophoresed in a direction perpendicular to the urea gradient. As the protein is electrophoresed, molecules at different positions across the gel are exposed to different urea concentrations. At positions where the urea concentration is high enough to promote unfolding, the mobility of the protein decreases because of the greater hydrodynamic volume of the unfolded form. When the gel is stained, or otherwise visualized, the electrophoretic pattern can be interpreted directly as an unfolding curve, as shown below for two forms of bacteriophage T4 lysozyme (adapted from Klemm et al. (1991)).
The upper band in the gel contains a mutant form of lysozyme (with Ala 160 replaced with Thr), and the discontinuous lower band contains the wild-type protein. The wild-type protein was applied to the gel first, electrophoresed for 10 min, and then the mutant protein was applied and electrophoresis was continued for an additional 20 min. The gel was stained with Coomassie blue. The gel reveals two important differences in the unfolding transitions of the wild-type and mutant proteins:
- The mutant protein unfolds at lower urea concentrations than the wild-type protein.
- The interconversion between the native and unfolded forms of the native protein is relatively slow (compared to the time of electrophoresis), giving rise to the discontinuity, while that for the mutant protein is quite fast, giving rise to a smooth protein band.
Although they generally provide only qualitative or semi-quantitative information, urea gradient gels offer several advantages for studying protein folding/unfolding transitions, since they are:
- Easy to implement, with only relatively inexpensive equipment.
- Require only small amounts of protein (typically 50 micrograms).
- Applicable to the analysis of complex mixtures.
- Able to detect conformational or covalent heterogeneity in protein samples.
Preparing Urea Gradient Gels
Although urea gradient gels are somewhat more difficult to prepare than ordinary SDS gels, for instance, the procedure can be readily mastered after a few tries. The slab gels are prepared between glass plates that are rotated 90 degrees from the orientation used for electrophoresis, so that the concentration gradient is initially vertical. This requires minor modification of most electrophoresis apparatus, including spacers to fit the sides of the glass plates (as oriented for casting) and some arrangement for holding the plates in this orientation.
The figure below shows an arrangement devised to prepare gels for the Bio-Rad Mini-Protean II apparatus.
The figure shows:
- A gel sandwich assembled and oriented for casting. Special spacers are designed to fit along the sides of the gel and keep the plates from sliding when placed in a casting box.
- The sandwich oriented for electrophoresis. The spacers used for casting have been removed, and two small short spacers have been placed to form a sample well above the upper surface.
- A casting box designed to hold the glass plates and casting spacers for five gels.
- The cover for the casting box, which is held on to the front of the box with binder clips. The inlet for the gel solutions is on the cover and directs the solution to the bottom of the V-shaped chamber below the glass plates.
The recommended procedure for preparing the gradients employs a three-channel peristaltic pump, as illustrated below. One channel of the pump is used to pump a solution containing 8 M urea into a mixing vessel that initially contains a solution with no urea. The other two channels pump the solution from the mixing vessel to the gel forms. As the solution is pumped from the mixing vessel and is partially replaced with the 8 M urea solution, the urea concentration increases linearly.
This figure is adapted from Goldenberg (1997).
In order to prevent polymerization before the gradient is fully formed, it is convenient to use a photo-activated polymerization catalyst. The gradients are prepared under low light (complete darkness is not required) and then polymerization is initiated by exposure to bright light. Traditionally, riboflavin has been used for photo-polymerizing polyacrylamide gels, but it is often difficult to obtain efficient polymerization with this catalyst, particularly at neutral or slightly alkaline pH values. More recently, methylene blue has been introduced as a photo-polymerization catalyst, and this reagent appears to be greatly superior to riboflavin, resulting in very efficient polymerization.
Because urea tends to retard the electrophoresis of proteins, even in the absence of unfolding, urea gradient gels are usually prepared with a compensating acrylamide gradient. For gels polymerized with riboflavin, the acrylamide concentrations in the 0 and 8 M urea solutions are 15% and 11%, respectively. When methylene blue is used, lower acrylamide concentrations, 7% and 11%, are recommended because of the higher efficiency of polymerization.
Additional details about preparing and using urea gradient gels are provided in the references listed below.
The original descriptions of urea gradient gels, including extensive discussion of the important experimental parameters and analysis of the relationships between transition rates and electrophoretic patterns:
- Creighton, T. E. (1979) Electrophoretic analysis of the unfolding of proteins by urea. J. Mol. Biol. 129, 235-264. http://dx.doi.org/10.1016/0022-2836(79)90279-1
- Creighton, T. E. (1980) Kinetic study of protein unfolding and refolding using urea gradient electrophoresis. J. Mol. Biol. 137, 61-80. http://dx.doi.org/10.1016/0022-2836(80)90157-6
Applications of urea gradient gels to the study of mutant proteins:
- Craig, S., Hollecker, M., Creighton, T. E. & Pain, R. H. (1985) Single amino acid mutations block a late step in the folding of beta-lactamase from staphylococcus aureus. J. Mol. Biol. 185, 681-687. http://dx.doi.org/10.1016/0022-2836(85)90053-1
- Klemm, J. D., Wozniak, J. A., Alber, T. & Goldenberg, D. P. (1991) Correlation between mutational destabilization of phage T4 lysozyme and increased unfolding rates. Biochemistry 30, 589-594. http://dx.doi.org/10.1021/bi00216a038
- Creighton, T. E. & Shortle, D. (1994) Electrophoretic characterization of the denatured states of staphylococcal nuclease. J. Mol. Biol. 242, 670-682. http://dx.doi.org/10.1006/jmbi.1994.1616
- Lyubimova, T., Caglio, S., Gelfi, C., Righetti, P. G. & Rabilloud, T. (1993) Photopolymerization of polyacrylamide gels with methylene blue. Electrophoresis 14, 40-50. http://dx.doi.org/10.1002/elps.1150140108
A general review of gel electrophoresis and applications to studies of protein folding:
- Goldenberg, D. P. & Creighton, T. E. (1984) Gel Electrophoresis in Studies of Protein Conformation and Folding. Anal. Biochem. 138, 1-18. http://dx.doi.org/10.1016/0003-2697%2884%2990761-9
Book chapters with detailed information about the practical aspects of urea gradient gel electrophoresis, including recipes and step-by-step instructions for preparing the gels:
- Goldenberg, D. P. (1996). Transverse urea-gradient gel electrophoresis. In Current Protocols in Protein Science (Coligan, J. E., Dunn, B., Ploegh, H. L., Speicher, D. W. & Wingfield, P. T., eds.), pp. 7.4.1 - 7.4.13. Wiley, New York.
- Goldenberg, D. P. (1997). Analysis of protein conformation by gel electrophoresis. In Protein Structure: A Practical Approach 2nd edit. (Creighton, T. E., ed.), pp. 187-218. IRL Press, Oxford.
The casting box and spacers illustrated above are available from:
Telephone: (631) 722-3635
FAX: (631)722-3106The spacers and casting box were designed to prepare gels for use with the Bio-Rad Mini-PROTEAN II gel apparatus. This apparatus have been replaced by a newer models. The gels prepared with the Aquebogue casting box and spacers should also fit the new model, though Bio-Rad is reluctant to offer a guarantee for this application, and I have not tried the new model. For more information, see the Bio-Rad Laboratories web site.