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)).

Picture of a urea gradient gel

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:

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:

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.

Picture of casting apparatus


The figure shows:
  1. 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.
  2. 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.
  3. A casting box designed to hold the glass plates and casting spacers for five gels.
  4. 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.
This figure is adapted from Goldenberg (1997). Drawings for the casting box and spacers can be downloaded here (pdf).

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.

Picture of method for casting gradient gels

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.

References

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:

Applications of urea gradient gels to the study of mutant proteins:

Photopolymerization using methylene blue:

A general review of gel electrophoresis and applications to studies of protein folding:

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:

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