Goldenberg and Creighfn (1984) introduced the use of polyacrylamide gels containing a concentration gradient of urea to analyze the folded state of proteins. A further description of this procedure, together with technical considerations and theoretical explanations, can be found in Goldenberg (1989). The following procedure has been refined from the literature of Goldenberg and Creighton (1984) and Goldetiberg (1989). This experiment was derived from the Laboratory Guide for Protein Purification and Characterization by Houzhu Zhu.
Operation method
Urea gradient polyacrylamide gel electrophoresis and folding analysis experiments
Materials and Instruments
Acrylamide Bisacrylamide Tris-acetic acid buffer Riboflavin Urea N N N' N'-Tetramethylethylenediamine (TEMED) Butanol Glycerol Bromophenol Blue Khomas Blue Dye Move Materials and equipment For more product details, please visit Aladdin Scientific website.
Peristaltic pump Peristaltic mixer with stirring platform Gel injection rack Gel injection slide Spacer Fluorescent lamp Plate gel electrophoresis device Power supply
Acrylamide
Bisacrylamide
Tris-acetic acid buffer (0.5 mol/L; pH 8.0)
Riboflavin (0.04mg/ml aqueous solution)
Urea (crystals; commercial ultrapure grade or deionized to remove cyanate)
Linear gradient mixer with stirring platform
N,N,N',N'-tetramethylethylenediamine (TEMED)
Peristaltic pump with silicone tubing
Flatbed injection rack
Plate for gluing (square plates only, not grooved plates with perforated combs)
Spacers' (two side spacers and two bottom spacers for standard glue)
Butanol, water-saturated
Fluorescent lamps
Glycerin
Bromophenol Blue (0.1mg/ml)
Plate gel electrophoresis device
Power supply (250V)
Kaumas Brilliant Blue Dye
Operating Procedures
1) Prepare the following gel solution:
N solution (15% acrylamide; no urea)
Acrylamide (30%; w/v)/bisacrylamide (0.8%; w/v) 50 ml
Tris-acetic acid buffer (0.5 mol/L; pH 8.0) 10ml
Riboflavin(0.04mg/ml) 12.5ml
H20 27.5ml
Total volume 100ml
Liquid D (11% acrylamide: 8 mol/L urea)
Acrylamide (30%; w/v)/bisacrylamide (0.8%; w/v) 36.7ml
Tris-acetic acid buffer (0.5 mol/L; pH 8,0) 10ml
Riboflavin(0.04mg/ml) 12.5ml
Urea 48.1g
H2O 4ml
Total volume 100ml
2) The gel solution was vacuum degassed in a flask with a side tube.
3) Place liquid N and liquid D in the appropriate position of the gradient mixer, the position depends on the direction of gel filling. If filling from the top, put liquid D in the front chamber (wordchamber), liquid N is the limiting liquid of the mixer (lmiitingsolution). If filling from the bottom, put liquid N in the front chamber, liquid D is the limiting liquid.
Note: Gradient mixer can be used with coaxial chamber type or parallel chamber type, but must pay attention to what kind of solution will be placed in the front chamber.
4) Fit the slide with the bottom spacer for the side spacers of a standard gel. They usually need to be adjusted so that they do not extend beyond the edge of the slide when the cast gel is turned sideways. Use standard side spacer strips for the bottom.
5) Stir the solutions in the gradient mixer. Add 100ul TEMEDd to each solution open the valve and turn on the peristaltic pump at a flow rate of about 1 ml/min. It is best to keep this setup in a cold room to avoid fluorescence induced polymerization.
Note: The volume may be increased or decreased to match the gel casting device.
6) Dump water saturated butanol to cover the gel and place directly under fluorescent light for 1 hour or until the gel sets.
7) Remove the water-saturated butanol and wash the surface of the gel with water. Insert a standard side spacer strip at the top of the gel. Rotate the gel 90°. Remove the spacer strips from the new top and bottom of the gel. A gradient of urea should now run across the gel from one side to the other.
8) Mix the sample (concentration at least 1mg/ml) 10:1 with glycerol and 0.1mg/ml bromophenol blue.
9)The gel was loaded into the power unit. Electrophoresis buffer was used with a 1:10-fold dilution of 0.5 mol/L Tris-acetic acid buffer (pH 8.0) (final concentration 50 mmol/L).
10) Add a layer of sample (50~100Ml) evenly on the top of the gel. Electrophoresis was started with a current of 10mA,flowing to the anode. Calmodulin is an anion and should move toward the anode at pH 8.0.
11) Remove the gel and stain with Caumas Brilliant Blue as described for standard Laemmli gels in Appendix 5.